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Manuel Varela: Jacques Monod, François Jacob and André Lwoff – Three who shared the Nobel Prize in 1965

Nov 9, 2017 by

An Interview with Manuel Varela: Jacques Monod, François Jacob and André Lwoff  – Three who shared the Nobel Prize in 1965

Michael F. Shaughnessy –

1) Professor Varela, often science and discoveries are the result of true collaboration.  It seems that Jacques Monod, Francois Jacob and Andre Lwoff had to work together in terms of their discoveries regarding the genetic control of first, enzyme and then virus synthesis.   First of all, can you give us a brief summary about these three scientists?

Dr. Shaughnessy, the trio of French scientists to whom you refer, Drs. Jacques Lucien Monod, François Jacob, and André Michael Lwoff, are early pioneers whose bacteriological and bacteriophage genetic experiments were of critical importance to the birth of the molecular biological fields of study. Together, the trio of scientific investigators Monod (a biochemist), Jacob (a biological naturalist and physician), and Lwoff (a microbiologist) received a share of the 1965 Nobel Prize, for their work in the categories of physiology or medicine.

Jacques Monod was born on the 9th day of February in the year 1910 in Paris, France. His parents were Lucien (father) and mother Charlotte (affectionately nicknamed ‘Sharlie’) MacGregor Todd. Lucien was French, and Sharlie was an American.  Accounts report that the Monod household was a center of intellectual and cultural interests, with emphases on the arts, music, and the sciences.  When Monod was a young child, the family moved to Cannes, where they lived a largely provincial life. Although Lucien was an artist, Jacques later attributed his early interest in biology directly to his father, who was an early fan of Charles Darwin and of evolution. Monod was enrolled in the College at Cannes (which later became a Lycée) until the year 1928 when he was 18 years of age. He was then enrolled at the Sorbonne, in Paris, where he majored in the field of natural sciences, namely, biology.  At the Sorbonne Monod took his undergraduate degree in 1931. After spending brief periods of study at the University of Strasbourg and University of Paris, in 1932 Monod became a lecturer of zoology. Encouraged by Boris Ephrussi to study genetics, Monod took the advice. He entered the famous research laboratory of Prof. Thomas Hunt Morgan, who was housed at the prestigious California Institute of Technology, where he was taught the fundamentals known in the field of genetics, at that time. Returning to the Sorbonne, Monod became a laboratory assistant in the research lab of Georges Teissier, and it is here where Monod became acquainted with bacteria. Interestingly, it is during this period of the Second World War, in 1940, when Monod had become an incognito member of the French resistance during the German occupation. In 1941, Monod took his doctorate in the natural sciences. He served as a research professor at the Sorbonne and later at the Pasteur Institute in Paris, where he ultimately became its director in 1971. Monod died of leukemia at the age of 66 years in 1976 on the 31st day of the month of May.

François Jacob was born on the 17th day of June in 1920 in Nancy, France, to parents Simon and Thérèse Franck Jacob, father and mother, respectively. In 1927, he enrolled at Carnot Lycée, located in Paris, where he was a pupil at the institution until 1937. In his autobiography, titled The Statue Within, Jacob related how he and his fellow students at the school were brutally traumatized by other students who were right-wing nationalists.  A family friend, a surgeon who let the young Jacob observe a surgical operation, inspired Jacob to become a physician. He entered medical school at the Faculty of Paris around 1938.  World War II disrupted Jacob’s medical education in the month of June of the year 1940 when he escaped to London by boat and joined the military.  He was initially stationed in Africa as a medical officer. He was then sent to Normandy where his arm and legs had been wounded severely. When the war ended, Jacob continued with his medical studies and earned his M.D. degree in 1947.  However, due to the nature and severity of his war injuries, Jacob was not able to perform surgery.  Thus, he turned his attention to research studies in biology. First, he worked to produce penicillin. Then he was invited by Dr. André Lwoff to join his research laboratory at the Institute of Pasteur, in Paris. In 1951, Jacob earned a degree in the sciences, and in 1954 he earned his doctorate of philosophy at the University of Paris Sorbonne. In 1956, he became a laboratory director at the Pasteur Institute. In 1960, Jacob became chair of the cell genetics department at the same institution. In 1964, he became professor of cell genetics at the prestigious Collége de France. On the 19th day of April in 2013, Jacob died at the age of 92 years.

André Lwoff was born in 1902 on the 8th day of May in Ainay-le-Château, Allier, France. His parents were Solomon and Marie Siminovitch Lwoff.  André’s father was a physician who knew the very famous Elie Metchnikoff, discoverer of phagocytes and first immunologist ever to win the Nobel. According to an elegant scientific biography of Lwoff written by his friend and colleague, Dr. Agnes Ullmann, the great Prof. Metchnikoff introduced a 13-year old Lwoff to the microscope. Lwoff was an undergraduate at the University of Paris, where he took his college degree in natural sciences in 1921. He had been an apprentice of sorts under the tutelage of another great scientist, Edouard Chatton, a protozoological taxonomist. At 20 years of age, Lwoff joined the laboratory of Felix Mesnil (a colleague of Dr. Pasteur) at the Pasteur Institute, in Paris, France, with the encouragement of Chatton. In 1927, Lwoff garnered an M.D. degree.  Dr. Lwoff then married Marguerite Bourdaleix, who would become an established investigator in her own right but unfortunately lived under the shadow of her husband.  In 1932, Lwoff earned his Ph.D. at the University of Paris. As a postdoctoral fellow at the University of Cambridge, the Rockefeller Foundation funded his work in the laboratory of David Keilin.  Next, Lwoff joined the Pasteur Institute in 1938. In 1959, he became professor of microbiology at the Sorbonne. In 1968, he retired from the Pasteur Institute and took on the directorship of the Cancer Research Institute, in Villejuif, France. In 1994, at the age of 92, he passed away on the 30th day of September.

2) Now , what exactly do we mean by virus synthesis?

Depending on your point of view, a mature virus, called a virion, may be considered amongst the tiniest of all living organisms or one of the most complex aggregates of non-living chemicals. Thus, if you hold that viruses are alive, then you might think that they are bona fide microbes who happen to need a lot of help from host cells to grow. On the other hand, if you hold that viruses are non-living, then you might think that viruses are simply virus particles. In any case, a virus consists of some sort of nucleic acid genome, like RNA or DNA, which is surround by a protein coat, called a capsid.  Some of the viruses have an additional biological membrane, called an envelope.

A key property of viruses is that they lack biological machinery to make more of themselves.  Thus, in order to propagate their numbers, viruses must gain access to the insides of cells, where biological machinery for nucleic acid and protein biosynthesis resides. In a technical sense, viral synthesis today refers to the stealing and using of internal cellular biological machinery in order to synthesize more viruses. We refer to biosynthesis as one of the steps in the infection process.

I think that prior to the 1950s, viral synthesis referred to the propagation, replication, or multiplication of viruses that occur during infection of a host cell by a virus. For our purpose of discussion here, there are two main types of viral infection to consider. Lwoff discovered one of these types of phage virus synthesis.

In general, virus synthesis occurring during infection of a host cell by a virion involves the following stages.  (a) The virus binding to a host cell receptor; (b) entry of the virus or its genome into the cell; (c) biosynthesis, involving nucleic acid genome replication, transcription to make RNA, and protein synthesis; (d) assembly of viral capsid and packaging of genome into the capsid; and (e) escape of amplified virus numbers from the host cell.

The trio Lwoff, Monod and Jacob studied a special type of virus that infects bacterial host cells.  These viruses are called bacteriophages or simply phages. Frederick Twort discovered the phages in 1915.

The phages and their host cells were quite easy to manipulate in the laboratory and served very well for the study of genetics. Although there are many types of viruses, I shall consider here primarily the phages.

The first type of viral infection was already known and has been called the lytic cycle, in which the host cell dies by being lysed—this cell lysis releases the multiplied viruses from the internally encased host cell. It allows the viruses to move about in order to seek new host cells to start the infection process all over again.

In the second case of viral infection, discovered by Dr. Lwoff, is called lysogeny. In this case, the infection starts out similarly to that of the lytic cycle, but the outcome of the infection takes a turn that differs from the stages outlined above. Lysogeny involves the following stages: (a) viral binding to the host cell; (b) viral or nucleic acid entry; (c) integration of viral nucleic acid into the host cell genome—at this point the virus is referred to as a prophage, and the bacterial host is called a lysogen; (d) binary fission growth of the bacterial lysogen—here, the lysogen can grow indefinitely if given the chance;  (e) induction of the lytic cycle—Lwoff discovered this by exposing the lysogen to UV light; (f) biosynthesis; (g) assembly & packaging; and (h) phage escape.

3) I have repeatedly heard about E. coli–   what exactly is it and why is it relevant to these scientists?

The term E. coli is the shortened version of the well-known bacterium, which, in its entirety, is officially called Escherichia coli.  Dr. Theodore Escherich, who had originally named the clinical isolate as Bacillus coli, discovered it.  It was later named E. coli in Dr. Escherich’s honor.

In general, there are two main categories of E. coli bacteria. The first kind is the pathogenic type, represented by a series of insidious strains of E. coli, all of which are causative agents of infectious disease, and some of which are quite severe.

The second type of E. coli is the non-pathogenic kind that is typically found to reside in the gut of all animals, including those of all humans. This relatively harmless kind of E. coli is often used as an indicator of fecal contamination in environmental locations outside of the research laboratory. Inside the research laboratory, however, it has been a different situation altogether. As a proverbial ‘laboratory rat’ of sorts, E. coli has been used for basic research, such as that seen in both early and now modern molecular biological research investigations.

In their Nobel Prize discoveries, the investigators Monod, Jacob, Lwoff and many others used E. coli as the host cells to study the regulation of gene expression, for the first time in history. They demonstrated the presence and activity of the so-called lac system, which they later called the lac operon.

4) Monod is apparently believed to be one of the founders of molecular biology. If so, where do the other two scientists fit in?

I think that both Drs. Lwoff and Jacob can be considered, with a very large degree of certainty, as bona fide co-founders of molecular biology, and on par with Dr. Monod.

As I mentioned above, Dr. Lwoff’s work was critical to the discovery of lysogeny. Starting in 1949, he had studied the phenomenon from the perspective of bacterial immunity to phage infection. Lwoff and his co-workers Niels Kjeldgaard and Louis Siminovitch worked with a bacterium called Bacillus megaterium because of its large size—which they could exploit to examine a single bacterial cell in the test tube and under a microscope.  Adding bacteriophage to his single cell, they could observe cell lysis resulting from the lytic cycle of phage infection. However, Lwoff and colleagues also observed a great many individual cells not undergoing the lysis and instead remaining intact. These lysogens stubbornly refused to be lysed.  Lwoff’s team tried every type of agent and failed to observe the lysis, until they tried, of all things, the UV light!  That is, UV light induced the lysogen to undergo the lytic phase of infection. Lwoff was to reminisce in later years that this finding was one of his greatest thrills of his investigative career in science.

Of the many discoveries made by Dr. Jacob, he had two quite notable ones.  The first one dealt with following up on Lwoff’s findings regarding lysogeny. Jacob and collaborator Elie Wollman focused on bacterial conjugation as a tool with which to study the lysogenic phenomenon using a bacteriophage virus called phage lambda (λ). They found that in the prophage stage of the phage λ, the viral genome integrated into the bacterial host genome, a phenomenon they called an episome (an integrated λ genome). The prophage episome formation occurred during bacterial conjugation.

The second key discovery involving Dr. Jacob involved his work with Dr. Monod and Dr. Arthur Pardee in which they discovered the famous lac operon. It was the first time in scientific history that the operon notion was examined and in such genetic detail. They had been interested in distinguishing between two seemingly contradictory hypotheses.  On the one hand, they wanted to know whether E. coli bacteria underwent induction of the enzymes that were needed to utilize the milk sugar lactose for energy and growth.  On the other hand, they also wanted to know how this so-called enzyme induction system related to the outwardly conflicting notion that end-products of enzymes inhibited the production of the enzymes that made the products in the first place, a situation known as repression.  In short, they wanted to reconcile the two hypotheses of enzyme induction versus enzyme repression with one grand unifying hypothesis.  In so doing, they discovered the lac operon system of gene regulation.

5) Monod and Jacob apparently first garnered fame and their reputation for their research on E.coli lac operon. What did this research show and why was it important?

The initial principle of the lac operon system first came to Jacob while watching a Western film with his wife Lise, during a vacation in August of 1958.

Jacob had thoughtfully compared his studies on the λ phage infection of B. megaterium to produce lysogeny with the studies pertaining to the utilization in E. coli of the sugar lactose for energy production and growth. Monod had just found E. coli mutants that had lost their ability to be induced to make lactose-utilizing machinery—that is, the mutants had lost inducibility and were thus referred to as i for loss of inducibility. These so-called i mutants (or lacI) made plenty of β-galactosidase enzyme, a genetic type called z+ (or lacZ+).

Jacob reasoned that the two systems, i.e., the λ-infected lysogen induction to commence the lytic cycle with UV light and the isolation of the so-called constitutive i mutants of E. coli, which showed loss of inducibility by continually producing the lactose metabolizing enzyme β-galactosidase, may be operating in much the same way. That is, the UV light may have broken a λ repressor to produce λ phage, and the mutation in the lac system may have involved a broken lactose repressor to produce β-galactosidase.  In essence, both seemingly different systems, i.e., the λ phage synthesis and the lactose utilization machinery, had a common type of molecular regulatory player: the repressor!

The story is told by Dr. Benno Müller-Hill in his delightful little book ‘The lac Operon’ that when Jacob first told Monod about this new insight (which was later demonstrated to be correct), Monod at first laughed and then ridiculed Jacob, explaining at length how it couldn’t possibly be correct.  The next day Monod was converted.

The development of the bacterial conjugation tool (that I mentioned above) by Jacob and Wollman, could be used by Jacob, Monod and Arthur Pardee to map the genomic locations of the genetic mutations by following the time needed for one donor cell to insert its genetic material into the recipient cell. At any point during the 100 minute conjugation period, the DNA transfer could be stopped simply by using a Waring blender (essentially of the same type used in any kitchen) to break apart the conjugating bacteria. In this way, Monod and Jacob could determine how closely linked the gene encoding the repressor protein (denoted LacI) was to the gene encoding the β-galactosidase enzyme (denoted LacZ). It was found that the lacI and lacZ genes were closely linked genetically. Using the conjugation method, it was also found that the y+ gene (lacY) encoding the lactose permease protein, LacY, was also closely linked to the lacZ gene.

The next experiment they did is well-known amongst molecular biologists and a widely celebrated one, which in turn led the way to the full discovery and elucidation of the very famous lac operon. This investigative project has been affectionately referred to as the so-called ‘PaJaMo’ experiment (or sometimes ‘PaJaMa’ but pronounced pajama or ‘pyama’).  The PaJaMo experiment was named as such after the first-two letters in the names of its experimenters, Pardee, Jacob and Monod.

The PaJaMo experiment involved conjugating a cell with a lacZ+ property (makes β-galactosidase enzyme) to transfer its z+ gene into a recipient cell that was both lacZ (could not produce β-galactosidase) and lacI (could make β-galactosidase because its repressor was broken).  The donor cell had a constitutive phenotype (made β-galactosidase with or without inducer) while the recipient had an inducible phenotype (made β-galactosidase only with inducer).  The conjugated cells made β-galactosidase regardless of whether inducer was present. Thus, the mated bacteria switched their biochemical behavior from a constitutive state to an inducible one!

Taking into account the rates of the β-galactosidase synthesis in the mated bacteria compared to the original parental generations, the concept of a repressor was formulated in which it (the repressor) could be induced to allow production of the β-galactosidase when in the presence of inducer. Monod, Jacob and Pardee reasoned that the DNA element to which the repressor acted upon was called the operator, or lacO.  Further mapping showed that the genes were arranged as follows: lacI, lacO, lacZ, lacY. Thus, the nature of the operon was established.

Further genetic and biochemical analyses showed that the lac operon worked in two distinctive modes: repressed versus induced.

The repressed mode was in operation when cells were grown without inducer called allo-lactose, a derivative of lactose. In this scenario, the continually made (i.e., constitutive) LacI repressor tightly binds to the operator DNA, preventing RNA polymerase from transcribing the lacZ and lacY genes to make RNA and thus preventing the synthesis of β-galactosidase and permease proteins.

In the induced mode, inducer (allo-lactose) is present and binds to the LacI repressor.  The inducer-repressor complex is unable to bind the operator.  Thus, in this scenario, RNA polymerase binds its promoter, turning on gene expression in the form of transcription and translation of LacZ and LacY.

With the notion of repression versus induction established with the famous lac operon, it paved the way for molecular biologists and biochemists to manipulate the gene expression programs for a variety of other genes.  Genes could be turned or off as desired. Such gene expression systems could be used for improvements in biomedicine. It led the way to the eventual development of gene therapies, production of important medicines, invention of critical diagnostic tools, and many other important applications.

6) François Jacob made some additional contributions in the realm of genetics by successfully working with other famous scientists like Monod and Lwoff while also working at the famous ‘Pasteur Institute’ of France.  What were some of these contributions?

While at the Institute of Pasteur, François Jacob was key to several important discoveries. He discovered many of the intricate mechanisms involved in lysogeny. He showed the inner workings of prophages and how they integrated as episomes into the chromosomes of host bacterial cells. Jacob helped to develop the bacterial conjugation method for the purpose of mapping mutated genes and studying their resultant behaviors within new host bacteria. His work led to our understanding of gene transfer between bacteria—a key feature now recognized in transfer of antibiotic resistance genes between different species of bacteria.

Jacob was a significant figure in the discovery of key mechanisms in the regulation of gene expression, using molecular players, like RNA as messengers, genes that are regulated, and genetic elements that group together several genes to mediate a particular behavior—that is, the notion of an operon. Lastly, while at the Pasteur Institute, Jacob also participated in the discovery of allostery, a phenomenon in which an effector acts upon an enzyme by binding to a site, which is not its active biochemical site, on the enzyme to alter its structure, resulting in its altered activity. Enzymes are often inhibited in this allosteric manner.

7) André Lwoff in 1954 began studying poliovirus. His experiments on the relationship between the temperature sensitivity of viral development and neuro-virulence prompted him to evaluate the problem of viral infection.  How did these investigations lead him to work with Monod and Jacob and how are they relevant?

When Dr. Lwoff began studying poliovirus he measured their virulence capabilities in neuro-tissue after repeated passaging in cultured host cells under varying degrees of temperature. Together with his wife Marguerite Bourdaleix Lwoff (a prominent research investigator in her own right), they found that the poliovirus managed to mutate into highly virulent forms, regardless of the temperatures used during the viral passaging in cells. Whether cold or hot, the new poliovirus variants demonstrated their profound virulence.

Analyzing the various conditions for mutant isolation, whether virological (such as bacteriophages or poliovirus) or bacteriological (such as lysogenic), all were shown to be useful in Lwoff’s collaborations with Monod and Jacob.  Such work led ultimately to the discovery of the mechanisms involved in the lytic versus the lysogeny cycles of phages and to the innate immune response factors directed against poliovirus.

8) I would be remiss if I did not mention Lwoff’s work on bacteriophages, microbiota and on the poliovirus.  First, can you explain what each of these are, and how they are relevant to medicine and perhaps to the Nobel Prize he won with Monod and Jacob?

Let us briefly consider first the bacteriophages. These agents are viruses that specifically infect bacteria. As mentioned above, Lwoff used phage λ to solve the mystery of bacterial immunity to phage infection. In so doing, he began the field devoted to the regulation of gene expression using the lambda repressor. Its molecular mechanism was elegantly told in a wonderful book called ‘The Genetic Switch’ by Mark Ptashne. As I mentioned above, Lwoff’s work was used to help Monod and Jacob to work on the famous lac operon. One application of bacteriophages involves an old idea but has recently been somewhat in vogue, and it is called phage therapy. The concept of phage therapy consists of using specific phages to treat bacterial infections.  Two recent developments have been published in which specific phages target listeria (Listeria monocytogenes) or the MRSA (methicillin resistant Staphylococcus aureus) bacteria.

Regarding the microbiota, it refers to the nature and composition of the microbes that reside in a particular environment, such as water, soil, or other organisms. Nowadays, modern microbiologists use the term microbiome to denote the species of microbes harbored in various environments. Lwoff’s work in this area was conducted in the 1930s when he had worked under Prof. Edouard Chatton at the Pasteur Institute and when Lwoff had had the Rockefeller grant to work in the laboratory of David Keilin at Cambridge. Under these venues, Lwoff studied various eukaryotic microorganisms such as the protozoan ciliates.  The human microbiome today has taken on monumental importance as being involved in many health-related issues, ranging from heart disease, diabetes, and cancer, to human emotion, such as happiness or moodiness.

It may interest you and our readers to know that poliovirus is very likely to go down in history as being the second microbe to be completely eradicated from the planet—smallpox virus was the first. Lwoff had had a hand in starting the polio eradication movement by studying how the innate immune system responds to poliovirus. When Lwoff showed that attenuated poliovirus mutants were temperature sensitive, Albert Sabin was able to exploit this knowledge to develop a viral marker for ensuring quality control during vaccine production.  Apparently, the marker test is still used to this day to test live oral poliovirus vaccine preparations.

9) What have I neglected to ask about this group of scientists and their collaborative effort?

I think it is fascinating that the collaborative works of Drs. Monod, Jacob, and Lwoff led to the establishment of many other fields of study within the domains of molecular biology, biochemistry, microbiology, and biomedical science. A brief listing is conveyed here.

First, their studies of microbial adaptation to sugar nutrients and the resulting microbial mobilization of the machinery required to metabolize the various sugars was seminal in the establishment of the microbial physiological field of study. Their study of enzymes like the LacZ β-galactosidase led to further work in protein chemistry, enzyme kinetics, and reporters for gene cloning and protein structure elucidation. Students of biochemistry and even microbiology laboratory courses may often still study the activities of LacZ.

The work dealing with the LacY permease led to groundbreaking studies of sugar transport across the membrane, transporter structure elucidation, energetic mechanisms of solute translocation, and substrate specificities. Incidentally, the trio had mistakenly predicted that the LacY permease was an enzyme, hence, the name permease.  The LacY protein is actually a transporter, rather than an enzyme, but the historical name ‘permease’ has stubbornly refused to go away, despite many attempts to correct it in the scientific literature.

The studies dealing with the LacI repressor have led to astonishingly significant advancements in protein homology structures, protein-DNA binding interactions, regulation of gene expression systems, gene cloning, and in gene therapy. This is a large field of scientific inquiry today involving many prominent investigators.

Their work addressing microbial genetics led to further work on gene mapping, bacterial and viral mutant isolation and mutant characterization, bacterial conjugation, and phage mechanisms of infection. In modern textbooks of biochemistry, cell biology, genetics, molecular biology, molecular medicine, virology, and microbiology, the lac operon and lysogeny are often given due prominence.

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