Avoid Boring People: Lessons From a Life in Science (31 page)

13. MANNERS REQUIRED FOR ACADEMIC CIVILITY

B
Y THE mid-1960s, more and more of the research being done in Wally's and my third-floor labs was directed toward understanding how gene functioning is regulated by specific environmental triggers. We were preoccupied by concepts emanating over the past decade from the Institut Pasteur in Paris. There Jacques Monod and Francois Jacob skillfully employed genetic analysis of the bacterium
E. coli
to study how its exposure to the sugar lactose induced the preferential synthesis of the lactose-degrading enzyme ß-galactosidase. They showed the existence of a lactose “repressor” whose presence negatively controls the rate at which ß-galactosidase molecules are made. Their work suggested that free lactose repressore bind to one or more regulatory regions on the ß-galactosidase gene, thereby preventing subsequent binding of the RNA-making enzyme RNA polymerase. In their 1961 Cold Spring Harbor Symposium paper, Jacob and Monod had proposed that the lactose repressor was an RNA molecule. Controversy by now existed as to whether they were correct, with others suspecting it to be a protein.

In 1965, Wally's main aim was to isolate the lactose repressor. As it was likely present only in a few molecules per bacterial cell, its identification was not a task for the faint-hearted. Two years before, Wally had spent several months unsuccessfully searching for it, believing it should specifically bind to ß-galactosidase inducers. Sensing then he was going nowhere, he turned to experiments with Julian Davies and Luigi Gorini that revealed streptomycin-induced misreadings of the genetic code, which offered possible explanations of how this powerful antibiotic kills bacteria.

Also keen to get the lactose repressor was the German biochemist Benno Müller-Hill, who was one year younger than Wally Coming from a politically liberal family, Benno gravitated further to the left as a chemistry student in the German socialist student scene, discovering that many teachers at the University of Munich had been Nazi sympathizers, though no one in authority seemed to care. In his hometown of Freiburg, Benno later did doctoral work in the laboratory of the sugar chemist Kurt Wallenfels. There he learned the essentials of protein chemistry through studying ß-galactosidase. He began to love science and became excited about Monod and Jacob's work on how lactose molecules induce the synthesis of ß-galactosidase. Later, in the fall of 1963, Benno began a postdoctoral position in Howard Ricken-berg's lab at Indiana University, to which he brought samples of the Wallenfels lab's glycosides to study their specificity in inducing ß-galactosidase.

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.