Brave Genius

by Sean B. Carroll

  FOR JACOB, THE lecture prompted him to be on the alert for results that might bear on understanding what was inside Crick’s black box. But as masterful as Crick’s presentation was, he did not address all of the fundamental mysteries surrounding genes and the control of protein synthesis. Crick’s primary interest, and that of a number of other molecular biologists, was the flow of genetic information from DNA to protein, and the nature of the genetic code. He made no mention of what had occupied Monod for years, which was the regulation of protein synthesis, the questions of how and why an enzyme was synthesized by bacteria in response to a nutrient—questions that now also engaged Jacob. Crick did not even cite a single piece of work from either of the Frenchmen.

  This was not a slight on Crick’s part. His focus was elsewhere. Crick was unquestionably the leader of the most elite circle within molecular biology. His lack of attention to the regulation of protein synthesis could be rationalized as him thinking “first things first.” It was reasonable to believe that the fundamentals of DNA, RNA, and proteins needed to be understood before much more complex matters, such as why certain proteins were made at some times and not others, could be tackled successfully.

  But science does not progress through central planning. At rare moments, an experiment may cast light where there was before only darkness. And in extremely rare instances, an experiment may fortuitously open the way to multiple discoveries. In one such experiment, Jacob and Monod would both break open the mystery of gene regulation and unexpectedly lead the way to discovering a key ingredient inside Crick’s black box.

  GENIUSES IN PAJAMAS

  The fundamental biological question posed by enzyme induction was: What was the “logic” that a simple, single-celled bacterium went through in order to make something (an enzyme) only when it could be utilized (when a certain sugar was present)? There were two approaches available to the puzzle: biochemistry and genetics. The thrust of the biochemical approach was to identify the molecules involved in the process, but this required one to sift through the thousands of different substances present in a cell and somehow fish out every component involved. This would be nearly impossible to do if one did not know how many and what kinds of molecules participate in a process.

  By contrast, in the genetic approach, the experimenter may identify through mutations all genes that affect a process. The power of the genetic approach is that it makes no assumptions about the number or type of substances involved. Each mutation, in effect, breaks or alters a piece of the machinery. The experimenter may then deduce how each component works by observing how each gene mutation affects the overall process or the activity of known components. The logic of the machinery may then be understood without having isolated any component in a test tube.

  By 1957, Monod and his team had identified mutations in two different genes that affected the ability of E. coli to synthesize ß-galactosidase. One type of mutation, called z–, eliminated the ability of the bacterium to produce an active enzyme. The other type, called i–, changed enzyme synthesis from being inducible to being produced all the time—or constitutively—regardless of whether an inducer was present. Monod and Jacob needed to better understand these mutations if they were to figure out how enzyme induction worked.

  It was a well-established test in genetics to determine whether a given mutation was recessive or dominant to a normal, unmutated version of a gene. Recessive meant that the trait was normal in the presence of one copy of the mutation, whereas dominant meant that the trait was altered by the presence of just one copy of the mutation. For example, albinism in humans and other mammals is recessive to other skin or coat colors. Plants and animals typically carry two copies of each gene and chromosome (except for the sex chromosomes in males). Monod and Jacob knew that they could better understand the effects of the z– and i– mutations if they tested them in the presence of one copy of normal, unmutated z and i genes (denoted z+ and i+). The hurdle for Jacob and Monod was that the E. coli bacterium naturally had only one copy of each gene in its single chromosome.

  But Jacob and Wollman, in developing their techniques for mating bacteria and mapping genes, knew how to make cells that contained two copies of a portion of a bacterial chromosome. Jacob and Monod drew up a scheme to transfer normal z+ and i+ genes into recipient bacteria carrying mutant z– and i– genes, and to determine whether the ß-galactosidase enzyme was produced.

  IN SEPTEMBER 1957, Arthur Pardee arrived from the University of California–Berkeley for a year’s sabbatical with Monod at the Pasteur. Pardee’s own laboratory had been working on the control of the synthesis of other enzymes. He had met Monod several years earlier, and subsequently heard enthusiastic reports about the atmosphere at the Pasteur from others who had visited.

  Pardee took on the challenge of carrying out the mating experiments that Jacob and Monod had designed. The novel idea was to measure enzyme synthesis in mated cells that carried various combinations of normal and mutated genes. Monod had constructed the necessary bacterial strains before Pardee arrived, but had hesitated to do the experiments himself because of various technical difficulties that he had anticipated. Monod, Jacob, and Pardee did not know at first whether enough cells would mate, and remain mated over the course of the experiment, such that they would be able to detect enzyme synthesis—if it indeed occurred.

  Pardee had to devise a series of tricks to make the experiments work. Some of his challenges were a matter of the differences in techniques and equipment between Berkeley and Paris. For example, Pardee wanted to maximize the amount of mating occurring in a mixture of the cells, but in order for the cells to be healthy over the course of the experiment, the cultures had to be well aerated. Usually, one would swirl the flasks vigorously on a rotating platform, but too much swirling disrupted mating. Pardee solved the aeration problem by putting only a small volume of liquid into a large flask, and swirling very gently. But when Jacob joined him in one experiment, he discovered that his French pipette was too short to reach the liquid in the flask. Pardee did not speak French well at all, but he did understand most of the profanity that Jacob uttered. They switched to using shorter flasks.

  With important technical details solved, Pardee set about testing what happened with various combinations of mutations and genes. Before conducting any series of experiments, Monod liked to map out the possible results and how each could be interpreted. Monod’s idea was that the i– mutation caused an inducer of the enzyme to be produced. If that was true, then he expected that transferring the z+ gene into a z– i– cell carrying both mutations would result in enzyme being synthesized, because the inducer present in the i– recipient cell would act on the transferred z+ gene.

  Pardee tried the experiment for the first time on December 3, 1957. It worked beautifully. Monod and Pardee were delighted to see that enzyme was produced within minutes of the transfer of the gene.

  As they pursued further experiments, however, they did not get the results Monod expected. When Pardee transferred genes in the other direction, moving the i– mutation into i+ z+ recipient cells that contained normal copies of both genes, Monod’s model predicted that enzyme synthesis would also take place; the inducer elicited by the i– mutation would cause the recipient cells to produce enzyme constitutively. This did not happen: no enzyme was produced.

  Moreover, as they repeated the original experiment of transferring the i+ and z+ genes into i– z– cells, they noticed that while enzyme synthesis always began promptly, it stopped within about two hours. Pardee and Monod were baffled.

  There were two possibilities: either the experimental design was flawed, or their ideas and therefore their expectations were wrong. The experimental controls indicated that the recipient cells were perfectly capable of making the enzyme when a sugar inducer was added to the culture. This result showed that the transferred z+ gene was intact and capable of functioning normally.

  Since the experiments were not deficient, then perhaps Monod’s ideas were.

/>   It would take a special visitor to help Monod see the error in his thinking. In the course of Pardee, Jacob, and Monod’s experiments, Leó Szilárd visited the Pasteur. The Hungarian-born physicist had turned to biology after the war and had been very busy as usual. Prior to arriving in Paris, he had just visited Crick’s group in Cambridge to discuss the genetic code.

  Those at the Pasteur looked forward to Szilárd’s visit and braced themselves for his tenacious but provocative questioning. His style was to pepper each scientist with questions and to write down the responses. Once finished with his interrogation, he would date the entry, hand the scientist his notebook, and say, “Sign there!” On future visits he would resume questioning by stating, “On such and such a date, you said this. Is that still true?”

  Szilárd was given an office in Monod’s laboratory for the duration of his stay, and he gave the customary seminar to the Pasteur group. Szilárd, too, had once worked on enzyme synthesis and stayed in touch with other scientists working in the field. In his seminar, Szilárd stressed an alternative logic controlling enzyme synthesis: instead of inducers working directly to activate enzyme synthesis, Szilárd argued that enzyme synthesis was normally inhibited or repressed, and that inducers acted by blocking that repression.

  Monod did not like the idea at all. He found it “repulsive” at first. The discussions with Szilárd in Monod’s office grew a bit tense at times. But as the experiments with Pardee unfolded, Monod realized that Szilárd was on the right track, and that he was not.

  The unexpected results forced Monod, Jacob, and Pardee to reconsider. After Szilárd had left Paris, Monod painted a new picture in a letter to Mel Cohn. He noted the “stupefaction” that he, Pardee, and Jacob had at seeing enzyme synthesis halt at two hours. He also told Cohn how when they inserted the i– gene into an i+ recipient, “one gets no synthesis of the enzyme at any time!!” Monod then offered a crucial deduction: “It is the gene i+ that is active and the gene i– (constitutive) that is inactive.”

  From these observations, Monod stated there were two possible interpretations. He led with what he felt was the “superior” hypothesis: the i gene determines the synthesis, not of an inducer, but of a repressor that blocks enzyme synthesis. Induction would then be due to the inhibition of the repressor by the inducer. The new model could then explain why enzyme synthesis shut down two hours after inserting the i+ gene—because of the accumulation of the repressor over time.

  Monod’s original logic had been inverted: enzyme induction occurred by the inhibition of repression, not simple activation. Repression did make logical sense: enzymes were not produced when not needed; they were repressed until an inducer de-repressed them.

  The flip in logic was a huge step. The repressor idea would be the cornerstone of a new logic of genetic programs. Pardee, Jacob, and Monod wrote a paper for the Comptes Rendus of the French Academy of Sciences. The experiments were named for the first two letters of each author’s last name, Pa-Ja-Mo; then the preferred moniker became the more memorable PaJaMa. Breaking open the logic of gene regulation was just one of two major dividends of the PaJaMa experiment; the very rapid synthesis of the enzyme in recipient cells was also a clue to Crick’s black box between DNA and protein. That dividend would take two more years to cash in.

  A MATTER OF HUMAN DIGNITY

  Pardee and Szilárd were just two of many scientists who were drawn to Monod’s clever band in Paris.

  While Agnes Ullmann had missed out on rubbing shoulders with Crick, Jacob, and others in England, she still wanted to use her passport to make contact with Western scientists. She decided that she would try to visit a place where she believed that there was interesting work going on. One of the few Western scientists she knew of was Jacques Monod—from both his scientific publications and his article on Lysenko in Combat that Györgi Adám had shown her many years earlier. She was determined to find some way to get to Paris to meet Monod.

  Getting from Budapest to Paris, let alone getting an appointment to see Monod, was complicated. She would need visas for each country through which she passed. Since she knew the French consul and scientific attaché, she had been able to get a transit visa to travel through France on her way to England. After that trip fell through, but with that paperwork in hand, she figured that she could visit France by taking a train through Austria and Switzerland. So she applied for and received Austrian and Swiss transit visas.

  Then there was the matter of paying for the ticket. She could not afford it. A number of colleagues from the time of the revolution paid her way.

  Tamás, now out of prison, was amazed that his wife even had a passport. Tamás predicted, “They won’t let you leave the country. They will stop you at the border.”

  Ullmann went nevertheless. She was allowed to carry just five dollars, so she smuggled another twenty dollars in a tube of toothpaste. Just in case she did make it out of Hungary, Tamás had alerted a cousin of his, Eva Ekvall, who lived in Paris.

  In early January 1958, Ullmann arrived at the Gare de l’Est railway station in Paris, and found Eva waiting for her with her name written on a piece of paper. Eva took Ullmann to the home she shared with her husband, an American military attaché, and gave her some more money. The couple was about to leave for the United States, so that very same day there was a big farewell party. Ullmann was very happy to enjoy the excellent buffet, but only for a while. When the Hungarian military attaché arrived at the reception, Eva told Ullmann to slip away because it would have been dangerous for her to meet him.

  Ullmann had not tried to write to Monod beforehand, so she still had to find a way to get an appointment at the Pasteur. Once in Paris, she tracked down Tamás’s uncle, who was also living in the capital. Fortunately for Ullmann, the uncle knew one of Monod’s cousins who had been in the Resistance with the scientist. The cousin agreed to phone Monod and to set up an appointment.

  The day Ullmann went to the Pasteur, she was very nervous. She waited in the office of Monod’s secretary, Madeleine Brunerie. She then heard a man come down the corridor whistling, perhaps a trombone theme from a Brahms concerto. He stopped in, and to her surprise, introduced himself, “I am Jacques Monod.” Ullmann introduced herself and explained that she had come to Paris hoping to work for perhaps six weeks in his lab.

  Pardee was also waiting for Monod, to show him the latest results in the PaJaMa experiments. Monod was clearly anxious to talk to Pardee, so he suggested to Ullmann that she give a seminar the next day about her doctoral work in Budapest.

  She came back the next day and talked to Monod’s group about her work on amylase synthesis in the pigeon pancreas. After the seminar, Monod asked Ullmann, “What are you doing in Paris and how long will you stay?” Monod had clearly forgotten about their conversation the day before.

  Ullmann, gathering all the courage she could muster, told Monod, “If you would allow me to work in your lab, I can arrange to stay six weeks. But if not, I will have to return to Budapest very soon.”

  Monod took Ullmann into his office and explained all of the work going on in the lab. He then asked her, “What would you like to do?”

  Ullmann said she would like to work with François Gros on the effect of antibiotics on protein synthesis in bacteria. She added cautiously, “If Monsieur Gros accepts me.”

  Monod burst out laughing, “François? He is the nicest man on earth; he never says no.” She started working with Gros the next morning.

  ULLMANN WAS THRILLED to be working with Gros and to be part of Monod’s group. As days went by, she began to divulge to Gros the troubles she had in Hungary—the crushing of the revolution, the crackdown on intellectuals, the arrests and trials of her colleagues, the secret police and Tamás’s arrest. The situation was unbearable and hopeless. She confessed to François, “I want to leave Hungary for good.” She added, “I want to come to France, and go on working forever in the lab.”

  Gros did not know quite what to say. He encouraged Ullmann to discuss her situati
on with Monod. Ullmann said that she would not dare do so. But Gros insisted, telling her that Monod “was the most understanding and nicest person on earth” and that if she was too scared to talk to Monod herself, he would do so on her behalf. Gros must have mentioned something to Monod, for the next day he invited Ullmann to dinner at his home.

  Ullmann went to Monod’s apartment on the avenue de la Bourdonnais. Monod’s brother, Philo, joined Odette and Jacques for the dinner. During the ’56 revolution, Philo had been working at the Ministry of Foreign Affairs and had helped Hungarian refugees who were stranded in Austria. The dinner conversation was dominated by talk of Hungary. Ullmann recounted all of her difficulties.

  After dinner, Monod, Philo, and Ullmann retired to a small salon for a smoke. Ullmann knew from Gros and from others in the lab that Monod had been in the Resistance. Yet she was still stunned when Monod told her that he would do everything he could to organize her and Tamás’s escape from Hungary. Monod needed to know what the consequences would be if she was caught. He asked, “What will happen to you if they pick you up?”

  “Twenty years in prison,” Ullmann replied.

  “So, do you accept this risk?” Monod asked.

  “Yes,” Ullmann said.

  Monod explained that it would take some effort to organize the plans. Ullmann asked her host, “Why would you help me?”

  “It is a question of human dignity,” Monod replied.

  At the end of the evening, Ullmann thanked Odette for the dinner and left with Philo, who took her back to the lab at the Pasteur, where she still had some late-night work to do. As they were riding in his car, Philo turned to her and said, “My poor child, you have never lived in a democracy, you don’t even know what it is.”

  After that momentous evening and hearing Ullmann’s eyewitness account of events in Hungary, Monod wanted her to have access to works that were banned in Hungary, especially those that were damning of Communism. Monod gave her Camus’s books; Trotsky’s autobiography, My Life; and Arthur Koestler’s Darkness at Noon. He also asked Gros to take Ullmann to see Camus’s play Caligula.