by James Watson
In Bloomington, Benno never felt comfortable either as a German among its many Jewish biochemists or as a leftist among Americans whose paranoia about communists in their midst surprised him. But his experiments there, which gave him sufficient results for a talk at the August 1964 International Biochemistry Congress in New York, were an ample reward for such social unease. By then he wanted to move on to the lactose repressor and came up to me after my talk to see whether I would accept him into my Harvard lab. Explaining that it was Gilbert he needed to approach, I urged him to visit Harvard as soon as Wally returned from a lengthy visit to England. Upon their meeting, Wally instantly saw Benno as the collaborator he needed and offered him a research position starting as soon as he could politely leave Howard Rickenberg's lab.
In Bloomington, Benno had learned how to genetically manipulate E. coli. He ably deployed this newly acquired skill soon after he arrived at Harvard to show that the lactose repressor is indeed a protein, not an RNA molecule. By using chemical mutagens he generated almost two hundred E. coli mutants that made ß-galactosidase in the absence of any inducers. Two of the mutants represented change to “nonsense” codons leading to premature polypeptide chain termination. If the repressor was made of RNA, this class of mutants would not have existed. The simple elegance of Benno's experiment was not revealed in his first manuscript draft. After telling him it was heavy and Teutonic, I rewrote it before its October 1965 submission to the Journal of Molecular Biology. As one of the journal's editors, I knew the article would quickly appear in print.
Though both Benno and Wally had earlier independently failed to detect the lac repressor through its binding to potent ß-galactosidase inducers, this feature offered still the only approach at their disposal. To increase their chances of succeeding, Benno again turned to bacterial genetics, making a mutant repressor that had enhanced affinity for the chemical that induced isopropyl-ß-D-i-thiogalactosidase (IPTG). By growing E. coli cells in very low concentrations of IPTG, a much more effective repressor became available. To double repressor numbers in bacteria, Benno made a diploid derivative containing two copies of its respective gene. These genetic tricks by themselves, however, were not sufficient to pinpoint the lac repressor in cell-free bacteria extracts. Success came only through developing molecular separation procedures that yielded protein samples enriched in the lac repressor. The first positive results were achieved in May 1966, but they were barely credible. Only 4 percent more radioactively labeled IPTG was found in bacterial extracts containing repressore than in surrounding repressor-free solutions. Soon better fractionation methods led to a semipurified sample that drew the IPTG into a semipermeable dialysis sac at a concentration almost twice that found outside. These enriched extracts were not affected by the enzymes that break down DNA and RNA. In contrast, the protein-degrading enzyme pronase destroyed all binding activity, confirming Benno's genetic pinpointing of the repressor as a protein.
Until then, Wally and Benno faced the likely prospect of not being first to characterize a repressore molecular nature. On the fourth floor was twenty-six-year-old Mark Ptashne, who was feverishly trying to isolate the phage λ repressor. It blocks the functioning of all but one phage λ gene when phage is present as an inactive prophage on an E. coli chromosome. The only λ gene then functioning is that coding for the repressor. Though its existence became known through elegant genetic experiments at the Institut Pasteur, no one in Paris had come up with a workable approach for its molecular characterization.
Mark had arrived in the fall of 1960 to do his Ph.D. thesis work with Matt Meselson. As essential to his nature as his desire to do top science were his leather motorcycle jacket, his violin, and his golf clubs. In high school, Mark had spent summer vacations at the University of Minnesota working in the neurophysiology lab of a left-wing family friend. At Reed College, which he chose over Harvard for its exclusive devotion to undergraduate education, he moved from philosophy to biology, working during the summer before his senior year at the University of Oregon. There Frank Stahl told him to do his graduate work with Matt Meselson. Mark already knew that the λ repressor was the next big objective in the phage world. But this goal was too risky for an early 1960s Ph.D. thesis, and so Mark settled in for a semiroutine genetic analysis of phage λ. As his thesis experiments neared their end, Paul Doty and I strongly backed his appointment to a three-year stint in Harvard's Society of Fellows. This would give him a shot at the λ repressor. As a candidate, he proved a shoo-in, since Wassily Leontief, the new head of the Society of Fellows, saw in Mark an agreeable conversationalist for the society's Monday night dinners. His term as a junior fellow commenced in July 1965.
That August, I submitted a $55,000 grant application to the National Science Foundation to pay Mark's salary and lab expenses for the three years, including a $5,000 yearly salary for a technician. The funding would allow him to work independently from his boss, Matt Meselson, who by now had despaired of Mark's sometimes sloppy work habits. In fact, my application stated that Mark intended to use DNA-RNA hybridization techniques to detect the λ repressor, which was yielding messy results even before the grant came through. Not enough was known about how RNA polymerase transcribes genes in cell-free extracts.
Mark's game plan soon changed. He began to look for differences in the proteins synthesized when heavily irradiated bacteria are infected with different types of λ phage. He guessed that λ repressor synthesis constituted only 0.01 percent of the protein synthesis in cells carrying λ prophages. To make scarce repressor molecules visible, he needed to drastically reduce synthesis of most bacterial proteins, as well as to inhibit the synthesis of all λ-specific proteins that were not the repressor. He reasoned he could cut back the routine synthesis of cellular proteins by irradiating the bacterial host cells with massive doses of ultraviolet light.
Mark Ptashne lobs a Softball at the 1968 Cold Spring Harbor symposium.
With Wally Gilbert's and my students and postdocs on the Biology Department rhino in 1965
Though Mark's experimental design was elegant, making it work would be no cakewalk. Though he received hints of early success, these were cruelly followed by failures to spot a radioactively labeled protein. In the summer of 1966, virtually all of Mark's experiments were crashing while Wally and Benno provided mounting proof that they were looking at the lac repressor. Happily, Mark's world would brighten immeasurably through the sudden unanticipated arrival of my former Radcliffe tutee, Nancy Haven Doe.
Nancy had been intrigued by repressore ever since learning about them during my spring 1963 Biology 2 lectures. Until then, she had expected her life to be largely that of the wife of a social male, very likely Brook Hopkins, Harvard ‘63, whom she had met as a freshman and with whom she had persevered through five years of “understood engagement.” During her senior year, noticing Nancy's intellectual vitality, I strongly encouraged her to go to graduate school. Aiming her toward the best, I wrote to Rockefeller University's president, Detlev Bronk, in support of her admission. Perhaps because she had come to science so recently, Bronk did not take the bait. Nancy's fate instead became Yale, possibly pushed ahead by my recommendation letter describing her as a quick learner who happened also to be cheerful and pretty.
Nancy's first year in New Haven was a typical full load of four courses during both the fall and spring terms. She mastered the Schrödinger wave equation as well as many facts of chemistry that with luck she would never need to use. Eight straight A's left Alan Garen no choice but to accept her into his molecular genetics lab, where she wanted to go for the repressor. Soon, however, she realized Alan to be a man of few words, little time for mentoring, and an excess of caution. He told her he was not up to the repressor; it was too hard a problem for someone over thirty-five. The dull alternative he proposed held little hope of sustaining and exciting her as a scientist. Writing to me early in March 1966, she remained resolute about resisting contentment in mediocrity. By late spring, she could take no more of her New Haven
abode and decamped with an equally disenchanted aspiring female academic to the island of Mykonos.
Upon coming back to the States, she feared that staying at Yale would condemn her to work in Bill Konigsberg's lab on dull, dull hemoglobin. In August, she wrote to me proposing to join Mark Ptashne's lab as his technician. There her three-and-a-half-year-long obsession to work on repressore could find a proper outlet. Only several days before, she'd visited Harvard and found Mark so clearly in need of intelligent help that he would forgive her several blessings of heredity including a six-inch advantage in height. Nancy found it infinitely more gratifying to work as a technician on the repressor than to be a graduate student not working on the repressor. Wanting my opinion about her potential new career, she made it clear she had not at any time been, nor ever would be, in love with Mark Ptashne. With that reassurance, I gave her my blessing.
Nancy nevertheless proved just the tonic Mark needed. With her methodically meticulous presence ensuring that he did not leave out essential experimental reagents, Mark could consistently detect the λ repressor and begin its molecular characterization. They soon showed that it was a protein of molecular weight near 30,000. Though Nancy at first believed Mark could win the repressor race, he knew otherwise. Wally and Benno were already writing up their paper, while he likely needed at least six more weeks in the lab before starting to write. Benno regularly came up to the fourth floor to check on their progress, much to Nancy's annoyance. She perceived his main purpose as gloating. Seeing Mark's always polite reception of Benno, however, Nancy managed an equal courage and grace. As for Wally, Mark so revered him that losing the race to him could never be devastating. Nancy's huge respect for Mark was no less evident: every day she went to the sandwich truck on Divinity Avenue to get his lunch, an egg salad sandwich, accompanied often by a chocolate eclair to fortify his morale.
Wally and Benno's paper “Isolation of the Lac Repressor” was submitted by me to the Proceedings of the National Academy of Science (PNAS) on October 24,1966, just in time for publication in the December issue. If I had delayed its submission to let Mark complete the experiments needed for his paper, Wally and Benno's discovery would bear the next year's publication date. Mark would have preferred this, but I argued that no one would deem his work less important for appearing in print second. On December 27, “Isolation of the λ Phage Repressor” went off to PNAS, to appear in the February 1967 issue. Mark triumphantly announced the λ repressor isolation at a seminar in our lab's tearoom a month before the paper came out. The room was packed and during his moment of triumph (“I did it all alone!”) he never acknowledged Nancy's key role in his success. Afterward, I badly chewed him out. Instantly realizing he had been too full of himself, that evening he called Nancy to apologize and later sent her flowers.
Mark and Nancy went on to test whether their repressor worked by binding to specific DNA sequences. When mixtures of radioactive λ repressor and λ DNA were centrifuged together in sucrose gradients they sedimented together. In contrast, mixtures of λ repressor and DNA from phage λ imm434 did not co-sediment. These much hoped for results were nonreproducible for an awful week until Mark realized that Nancy had inadvertently raised the salt levels in their mixtures. She nervously repeated the experiment using the original salt levels. The new results were just coming off the isotope counter when Nancy and Mark had to attend a seminar on the floor below. Halfway through the talk, the suspense overwhelmed her and she returned to the lab, where she quickly realized all was again well. She and Mark were for the first time ahead of Wally and Benno. Returning from the seminar, Mark shouted for joy, and they went into the halls to spread their good news. Going downstairs, they caught Wally and me about to leave the Biolabs. Upon learning that the experiment was repeatable, Wally's face turned ashen. Mark's overtaking him was not acceptable.
Over the weekend, Wally set about to do the analogous experiment using DNA from a phage Benno obtained from Jon Beckwith of Harvard Medical School that carried the ß-galactosidase gene and its control region. By Monday morning, Wally let it be known that he had preliminary positive results he intended to verify quickly, bringing him again even with Mark and Nancy. Seeing Wally's intense competitiveness made Nancy feel as if she could never survive the male-dominated dog-eat-dog grind of being a scientist. It also was an eye-opener for Mark. His reverence for Wally had taken a big knock.
But Wally's expectation of catching up to Mark and Nancy hit some skids. Holding him up were difficulties in purifying the radioactively labeled lac repressor. Though Mark gallantly held up his submission to Nature, intending to let Wally and Benno publish simultaneously, after two months they were not ready even to start writing, so he sent his paper in without theirs. Having been forewarned about Mark's submission, Nature's editor, John Maddox, sent it to the printer the day it arrived. “Specific Binding of λ Phage Repressor to λ DNA” appeared on April 15,1967, only six days after arriving in Nature's London office. Wally and Benno's paper “The Lac Operator Is DNA” was submitted on October 28 to PNAS, in time at least for publication in the same calendar year.
Nancy became a graduate student again in the fall of 1967 and continued to work in Mark's lab. I had told her to apply to Harvard and a week later she was formally accepted. Mark also encouraged her, saying she was brighter than any of the relatively few women and more than half the men at Harvard. She glowed in the knowledge of the approval of someone she revered. After the misery of Yale, everything had worked out, including her engagement to Brook: soon after becoming Mark's technician she had also become Nancy Hopkins. They moved to Trowbridge Street, their apartment graced by my wedding present of a life-size leather pig, similar to one Brook had long admired in his exclusive “final club” on Massachusetts Avenue.
After his second paper came out, Mark devoted more time to being a visible leftist, joining several South American neurophysiologists in Havana, where he met the cartoonist Jules Pfeiffer. Later he would go to Hanoi and Saigon on a trip organized by a former junior fellow, the MIT linguist Noam Chomsky. Over the past year, our tearoom conversations had centered more on Vietnam than on science, and I remained glued to an issue of the New York Times covering the Tet offensive all through a talk by MIT's Ethan Signer on bacterial-gene-carrying λ phages.
Earlier Benno and Jon Beckwith had marched down Massachusetts Avenue from Cambridge to Boston to demonstrate against America's Southeast Asia policy, discussing λ phages along the route. Immediately following Tet, Benno went with his soon-to-be wife, Barbara, to New York City for a big antiwar rally in Central Park. Equally anti-establishment in outlook, Barbara had first met Benno at one of Mark's parties in his flat north of the Biolabs, on Sacramento Street. Mark's blond-haired, live-in girlfriend, Micky, was from an even more leftist, if not Stalinist, background. Mark frequently escaped with his violin to their cottage on Cape Cod for weekends of heavy fellow-traveler chitchat.
In the summer of 1967, Wally and Celia Gilbert's three-year-old daughter, Elsbeth, was diagnosed with an incurable metastatic sarcoma. They made constant visits to Children's Hospital, compounding Wally's exhaustion when he got back home from late evenings in the lab. His two other children, John and Kate, earlier had thrilled to their father's eureka at finding the repressor. Now the mood of the house on Upland Avenue was grim. Hoping to distract them, in mid-fall I persuaded an ABC-TV producer, Ernie Pendrell, to feature Wally and Mark on a forthcoming TV special about science. Initially he had wanted to do an hourlong feature about my work, sponsored by North American Rockwell. But I told him the race for the repressor would show science at its brightest, making the public aware that ambitious young scientists, like young poets, are more creative when not encumbered by the braking powers of maturity, which I already felt nipping at my own heels. With Harvard's approval, an ABC crew came during the second week of February 1968 and followed me and Wally walking in Harvard Yard with Wally's children eagerly asking on cue, “Did you find it yet, Daddy?” A week later, the crew returned for a
raucous Lincoln's Birthday party I threw in my Appian Way flat.
By then, Mark was secure in the knowledge that as of July 1, he would be a member of the Harvard faculty. By appointing him a lecturer in biochemistry instead of an assistant professor, the University could match the salary recently offered him to be an associate professor at Berkeley. In support of his appointment, Francois Jacob wrote to Paul Doty that “Mark is the most gifted young man in his generation of biologists.” Franklin Ford orchestrated swift movement by the administration. The day after Christmas he wrote to tell me Ptashne was confirmed in his decision to stay at Harvard. Several months after, when Mark's appointment formally began, the Harvard Council of Deans honored him and Wally with the prestigious Ledlie Award for 1968, which included a $1,600 honorarium.
