Origins of Molecular Biology: A Tribute to Jacques Monod, Revised Edition

Madeleine Jolit provides information on her experience in lab. The author and her group were isolating mutants and carrying out the first assays of amylomaltase and galactosidase. Jacques Monod invented the “bactogène,” an apparatus designed to maintain “a continuous process for the cultivation of microorganisms, involving continuous and simultaneous addition of nutrient medium into, and removal of culture liquid from a fermenter." Monod was busy setting up his new service, welcoming and taking care of his students both in the lab and at the university. He would work only occasionally at the bench now, participating in some permease assays, having a look at the petri dishes. The author is thankful to this unique atmosphere which everyone in her group had created. She had known many labs in France and abroad, but had never seen anything like the one she had worked at. In that sense, she and her group had been very lucky.

In this chapter, the author relates his experiences as Jacques assistant at the laboratory. The core of the question in those days (1948–1950) was to understand why there was an increased rate of enzyme formation upon addition of the substrate (adaptation) or a diauxic inhibition. The working hypotheses in the lab were that “many different enzymes may stem from a common precursor or pool of precursor molecules” and that the “master pattern configuration determining the specificity was not the enzyme itself but a pre-existing self-duplicating unit (the gene).” On April 11, 1949, the author and Jacques spent the day at 37№C to give birth to the “bactogène.” The results were encouraging and Jacques wrote in his notebook the theory which was the basis of the bactogène.

On arriving at the Pasteur Institute, the author told Jacques Monod that he had come there to learn about adaptive enzymes and that he wished him to suggest a problem for him to work on. As a matter of fact, a strong candidate for a “preenzyme” had already been found in noninduced E. coli by Mel Cohn and Annamaria Torriani. Cohn and Torriani had shown that there was present in extracts from noninduced cells an enzymatically inactive protein which they called Pz. This protein cross-reacted with antibodies raised in rabbits by immunization with purified β-galactosidase (Gz). The experiment that Jacques proposed was indeed very simple. He had recently received a series of twenty auxotrophic mutants of E. coli from Bernard Davis, each requiring a different amino acid or accessory factor for growth. Enzyme synthesis began almost immediately upon adding back the required amino acid or growth factor. At this time, the kinetics of enzyme production were being followed as a function of time. It seemed to the author that if growth were indeed required, enzyme production should be measured with respect to the increase in bacterial mass. The final and complete elimination of the Pz protein as a precursor having anything to do with induction of β-galactosidase synthesis came a few years later when Cohn, Lennox, and Spiegelman transduced the lacZ gene into an i+z- Shigella dysenteriae containing no Pz, and obtained lac+ recombinant Shigella.

After the liberation, in 1945, Jacques Monod joined André Lwoff’s laboratory at the Pasteur Institute. The organizers of a symposium had asked the author to trace in a personal way the contributions of Monod to the origins of their present concept of induced enzyme synthesis. The key to the power of these Monod theories, 1947, 1961, or 1965, was simply that they were physiological-level theories capable of reductionism; that is to say, they were capable of an analysis at the level of chemistry. The author believes that Monod had one of the most creative minds of our time because he had one of the most creative minds simply because he thought deeply, ascetically, about how knowledge is acquired; and it is this process that he insisted should be the only basis for a system of ethical and aesthetic values.

The most promising approach to the study of the lactose system appeared to lie through genetic analysis. Conjugation had become a useful instrument for the analysis of any bacterial function. Starting from the viewpoint of the phage system, the author wanted Jacques’ opinion about the implications for the lac system of two basic concepts: (1) Repression (or induction) operates not progressively, but like a switch, by a yes-or-no, an on-or-off mechanism that involves only two states; and (2) Genetic units of an order higher than the gene must exist: “units of activity” that contain several genes subject to unitary expression, such expression probably being regulated at the level of DNA. Actually," Jacques concluded, "there is no direct evidence either for or against the idea of repression at the level of DNA, and we should keep this possibility in mind." His objection to the switch, the on-or-off concept of protein synthesis, appeared more serious to the author. During a small meeting in Sydney Brenner's room at King's College, the author described the latest results obtained in Paris and Berkeley on the regulation of protein synthesis and mentioned once again the unstable RNA hypothesis. The whole system could be viewed as an on-or-off switch alternating between two states, just like the switch of the small electric train. In the fall of 1960, Jacques and the author decided to assemble the various pieces of information then available into a story, mainly written by Jacques.

The epoch, that of molecular biology, may in its turn be seen as a composition of various “scenes,” as those which are threaded along a play by Bertolt Brecht, where a multitude of protagonists move about and spread their energy with passion. Jacques Monod has been, and will be remembered as, one of those very great men around whom the main events of contemporary biology have synthesized and harmoniously gathered, as the large tableaux around Brecht’s hero. According to the author, the history of RNA is that of a mysterious substance which did not interest anybody except a few cytologists. The finding that plant viruses contain, apart from proteins, ribonucleic acid has not only led one to perceive the functional universality of the RNA in biological systems, but has also helped in the analysis of its physicochemical composition. Jacob, Meselson, and Brenner brilliantly demonstrated that Volkin and Astrachan's RNA operated on preexisting ribosomes as a template biopolymer, organizing as a real "viral messenger" the proteins newly synthesized by the phage. The birth, at times difficult, of messenger RNA was received with great acclaim. The finding of a strict complementarity in the sequences of the messenger RNA and the homospecific DNA (Spiegelman) was also a major contribution.

The two-month lab course designed by Jacques Monod in 1956 and 1957 was intended to breach the gap between university teaching and living research. It was the author's real introduction to what was to become molecular biology—covering DNA, proteins, physiology, and biochemistry of bacteria and phages. The study of cross-reacting material (CRM) was of course prompted by the need to map the structural gene for galactosidase so it could be distinguished from genes involved in induction. But it was also to understand the obscure relation between that old ghost Pz and CRM of galactosidase. A scheme of competition between galactosidase and CRM was devised in which the only thing measured was enzyme activity. The is mutant which became a cornerstone of the theory of induction by negative control appeared by luck. It was a strange lac negative, giving numerous revertants which were all constitutive. It proved to be trans-dominant and fit nicely with the theory.

During the course of an analysis of the ability of compounds related to lactose to act as inducers of β-galactosidase, Howard Rickenberg, working with Georges Cohen and Gérard Buttin in Monod’s laboratory, discovered the inducible transport system (“permease”) for β-galactosidases. Monod's earlier studies had shown that the genetic regulation of the galactoside permease was coordinated with that of the β-galactosidase, and indeed that the gene for β-galactosidase (the z gene) and that for the permease (the y gene) were part of the same genetic unit, or operon. Monod proposed that the permease would have the property of binding its substrate and suggested that the author's group try to identify it by this property. The active galactoside permease has not yet been isolated, but Fox and Kennedy have isolated and characterized the y gene product, which they called the M-protein, after modifying it with the N-ethylmaleimide, using a clever modification of the binding idea based on Adam Kepes' observation that the β-galactosidase permease contains essential sulfhydryl groups that are protected by the substrate. The exit of galactose was also found to be inhibited by substances, such as α-methyglucoside and succinate that were not substrates for the galactose permease. Monod was intimately involved in everything that was going on in the laboratory, from sporulation to permeation, and in those days he was always in the laboratory, available for discussion of the work.

The enzymological and purification data on acetylase, pointed out some of the difficulties in believing that acetylase was permease or part of it, but left it an open question. One of the first things to do was to get acetylase in pure form, find out how much was made in the cell, and measure its size. The surprise from the purification data was that there was very much less acetylase produced than β-galactosidase, 10 to 35 times less by weight, depending on conditions of growth. Acetylase was fairly straightforward. By physical and chemical studies it was clearly a dimer of two identical chains, each of about 30,000 daltons. Antibody to acetylase did not cross-react with β-galactosidase, nor with anything else in the cell. The subunit structure of β-galactosidase was a difficult problem. But when end-group and other studies were carried out, it became clear that β-galactosidase contains long, not short chains. In fact, the polypeptide contains 1,021 amino acids. β-Galactosidase has been a challenge, worthy of a lot of work. It still hasn’t lost its interest. There were and still are interesting mutants, as well as the structure to wonder about. Though acetylase was and is revisited from time to time, β-galactosidase has been the focus of attention in the author's lab for many years.

Enzymes involved in the degradation of substrates similar in structure seemed likely to derive from the same precursor. For this reason Monod decided to study the enzymes involved in the degradation of two similar disaccharides: lactose and maltose. As a result, two communications were presented on July 12, 1948, at the French Academy of Sciences. One, signed by J. Monod, A. M. Torriani, and J. Gribetz, described the occurrence, in Escherichia coli, of lactase, only present in lactose-grown cells. In the other, signed by J. Monod and A. M. Torriani, was reported the existence of an "amylomaltase," present exclusively in maltose-grown cells. Because the "lactase" was in fact a β-galactosidase of broad specificity, it became possible to synthesize a chromogenic substrate for this enzyme, and therefore to render its assay both very simple and very sensitive, while the assay of amylomaltase still required the use of the cumbersome Warburg apparatus. The corresponding mutations generally mapped in the vicinity of the amylomaltase-phosphorylase operon. Other mutations, mapping in this second region, led to the most incredible phenotypes. The unwarranted complexity of the two most well-known positively regulated systems made Monod, and many others, suspicious that something basic could be wrong. Still, over the years the author could not escape imagining what would have happened if Monod had focused on amylomaltase, rather than on β-galactosidase.

At the meeting of the author and Jacques Monod, Monod explained to him how he intended to organize his biological training. By deciding that his essay should bear on "prediction of the conformation of a protein from its amino acid sequence," Monod put the author right into the field of protein folding, a theme which has since remained the subject of his research. When, after a decade, the author tries to understand why he got so deeply involved in that subject, he can find two main reasons, both related to the fact that Monsieur Monod himself had become particularly interested in protein folding. To account for the large size of proteins, Monod tried to analyze the constraints which the functional features of a protein exert on its structure: the existence of a catalytic site, requiring the exact relative positioning of half a dozen amino acids; the stability of the protein conformation, requiring, according to the “oil drop model,” a hydrophobic core; the solubility of the molecule, requiring the presence on its surface of polar residues that are able to interact with the solvent. Monod's interest in protein conformation survived his involvement in the direction of the Pasteur Institute.

The author's plan was to learn phage genetics for use in later physicochemical studies of genetic problems. Two surprises were awaiting him. One was a student (Alex Fritsch) who wanted to work with him; he was particularly interested in the uses of the analytical ultracentrifuge. The second surprise was a manuscript by Jacques Monod on a model for allosteric enzymes. It proposed a general model for allosteric enzymes. The particular protein which was discussed in detail was hemoglobin, which was not then known to be an allosteric protein. He looked down at the set of four dice, glued together, which he used as a model in thinking about the symmetry properties of oligomeric proteins. But the logic of the allosteric model seemed to require that proteins be flexible. Jacques admitted then that the answers to allosteric mechanisms would probably come from physicochemical studies of purified proteins, together with their crystal structures. Hemoglobin, in particular, became one of the most intensively studied molecules known to chemists.

This chapter focuses on mother nature and the design of a regulatory enzyme. Jacques Monod was looking at a nascent science from his peculiar point of view, molecular evolution. Each new result was presented in the lecture as one tiny piece in the puzzle that Mother Nature had solved. Many scientists appreciate the Monod–Wyman–Changeux model (M–W–C model) because it is a simple, elegant, and imaginative proposal. The author feels sad that a very significant confrontation between two different approaches of molecular biology is reduced to a formal conflict between two hypothetical kinetic pathways. He thinks that what is really at stake is a triple issue. First, it is a good example of classical opposition between informative and selective theories in biology (in the M–W–C model, preexisting states are selected by small metabolites, the concentrations of which reflect the various physiological needs of the cell; according to Koshland and his collaborators, the ligand informs the protein structure and directs its conformational change). Second, they diverge on the basic unity or on the diversity of the structural solutions historically retained by evolution to solve a problem of regulation. Third, Monod’s theory is falsifiable: to refute it does not simply mean to show how significantly the real solutions differ from a model. In Monod, as a public man, the author found the basic need for creative freedom.

The author thinks his first meeting with Jacques Monod was in the mid-1950s, probably after Jim Watson, and he had put forward the DNA structure. He clearly recalls giving a seminar at the Pasteur Institute in which he suggested the quite erroneous theory that during protein synthesis the inducer was needed to fold up the enzyme correctly. Monod was an excellent sailor who could manage his 37-foot sloop by himself, whereas he was always a rather bumbling amateur. One year there was a scientific meeting at Naples. This chapter discusses the author`s most rewarding sailing experience with Jacques. Jacques was very companionable—one always enjoyed an evening when he was there—and it’s not everyone that one can go sailing with. Their general attitude to most scientific matters was very similar, yet their backgrounds were sufficiently different to make both of them eager to hear what the other thought. As each new thing comes up, one regrets so much not being able to talk it over with Jacques. Jacques would understand so quickly; he would appreciate the importance of the point; he would say something illuminating that hadn’t occurred to one; one could reach agreement together and a deeper understanding. It is for this reason that the author finds that he has no stomach for writing a book about Jacques` work even though the author still feels a small nagging sense of duty.

The author recalls a visit of Jacques to Urbana in the early 1950s, in the course of which they talked of enzyme induction and of host-induced phage modification. The author's experiments at that time dealt with the expression of phage-transduced lactose genes. It was a well guarded secret that similar experiments on lactose-transferring episomes were underway in the laboratory, the experiments which later led to the formulation of the operon theory. In his last years Monod became concerned with the impact of biology on human society, and, characteristically, tried actively to promote the field of bioanthropology. The author believes Monod was inclined to put more confidence in the ethological and sociobiological approaches than the author thought was warranted. However, Monod was not dogmatically “biologizing” the human predicament, but simply trying to choose and develop a feasible approach.

In the course of research work, the author's group observed a fact that seemed very significant. A certain compound, phenyl-β-D-thiogalactoside, devoid of inductive capacity, proved capable of counteracting the action of an effective inducer such as methyl-β-D-thiogalactoside. The study of galactoside permease was to reveal another fact of great significance. Several years earlier, following Lederberg’s work, the group had isolated some “constitutive” mutants of β-galactosidase, that is, strains in which the enzyme was synthesized in the absence of any galactoside. The author then had to admit that a constitutive mutation, although very strongly linked to the loci governing galactosidase, galactoside permease, and transacetylase, had taken place in a gene (i) distinct from the other three (z, y, and Ac), and that the relationship of this gene to the three proteins violated the postulate of Beadle and Tatum. The model that the group had studied is interesting primarily because it proposes a functional correlation between certain elements of the molecular structure of proteins and certain of their physiologic properties, specifically those that are significant at the level of integration, of dynamic organization, of metabolism. If the proposed correlation is experimentally verified, then it is an additional reason for having confidence in the development of our discipline which, transcending its original domain, the chemistry of heredity, today is oriented toward the analysis of the more complex biological phenomena: the development of higher organisms and the operation of their networks of functional coordinations.