Tag Archives: James Watson

Wendell Stanley: First to Crystallize a Virus

In 1935 Wendell Stanley crystallized tobacco mosaic virus (TMV); an accomplishment for which he was awarded a share of the 1946 Nobel Prize in Chemistry. As a matter of history, Stanley’s Nobel award was the first ever bestowed on a virologist.

Wendel Stanley. 1946 Nobel Prize photo.
Wendel Stanley. 1946 Nobel Prize photo.

I have long considered Stanley’s achievement to be one of the most important developments in virology and, indeed, in biology in general. To appreciate why Stanley’s feat might have been so significant, we need to consider how little was known in the mid 1930s—and for the next two decades as well—about the chemical nature of genes. In fact, it was still widely assumed that genes are comprised of proteins. That was so because before James Watson and Francis Crick solved the structure of DNA in 1953, that molecule was thought to be structurally simple; rather like a starch. In contrast, proteins were structurally complex, and their wide variety seemed to provide for a virtually unlimited number of genes. But, as you might suppose, attempts to explain how proteins might be replicated led to rather unsatisfying models. Consequently, many serious biologists of the day adhered to the vitalist belief that life could not be explained by known laws of physics and chemistry.

The 1928 experiments of Frederick Griffith, involving the bacterium Diplococcus pneumoniae, provided the groundwork for later experiments by others that would cause some to consider that DNA indeed might be the genetic material. Griffith demonstrated that exposing live avirulent pneumococcal cells to an extract prepared from heat-killed virulent cells could transform the avirulent cells into virulent ones. [These experiments were part of Griffith’s efforts to create a vaccine against D. pneumoniae. He died in 1941, never knowing that his work would constitute one of the keystones of molecular biology.]

Griffith’s 1928 experiments were followed by the 1944 experiments of Oswald Avery, Colin MacLeod, and Maclyn McCarty, who identified the transforming activity in the extracts of virulent pneumococcal cells. Avery, MacLeod, and McCarty fractionated the extract into its various macromolecular constituents—protein, lipid, polysaccharide, and DNA. Next, they asked which of these fractions might have transforming activity. To the surprise of almost everyone, only the DNA fraction transformed avirulent, non-encapsulated pneumococcal cells into virulent encapsulated ones.

The remarkable findings from these transformation experiments were met with widespread skepticism. That was so because it was difficult for the classically trained geneticists of the day to accept these strange, seemingly bizarre experiments. Classical geneticists experimented by crossing organisms; not by transforming them with extracts. What’s more, they thought in terms of hereditary units called “genes,” rather than in terms of molecules of nucleic acid, or whatever other substance that genes might be comprised of. Moreover, as noted above, DNA was viewed as a rather uninteresting molecule. Thus, most biologists of the day continued to hold the view that genes are comprised of protein.

As to the state of virology in the mid 1930s, most interest in the field was concerned with medical and agricultural issues. Moreover, essentially all that was known about viruses per se was that they are smaller than bacteria and can propagate only within suitable host cells. Thus, virology had not yet advanced biological knowledge in general (see Aside 1.)

[Aside 1: Stanley relates in his 1946 Nobel lecture (1), “…when the work on viruses, which is recognized by the1946 Nobel Prize for Chemistry, was started in 1932, the true nature of viruses was a complete mystery. It was not known whether they were inorganic, carbohydrate, hydrocarbon, lipid, protein or organismal in nature. It became necessary, therefore, to conduct experiments which would yield information of a definite nature. Tobacco mosaic virus was selected for these initial experiments because it appeared to provide several unusual advantages…”]

Nonetheless, by the mid 1930s biochemists had made great strides in purifying and crystallizing proteins. [Solving the structure of proteins by crystallography was still well beyond the technology of the day.] Inspired by the success of the protein crystallographers, and encouraged by his evidence that TMV is at least partly a protein, Stanley proceeded to crystallize TMV (see Asides 2 and 3).

[Aside 2: Stanley’s evidence that viruses are comprised of protein was recounted in his Nobel lecture (1): “…in studies with pepsin it was found that this enzyme inactivated the virus only under conditions under which pepsin is active as a proteolytic agent… It was concluded in 1934 that “the virus of tobacco mosaic is a protein, or very closely associated with a protein, which may be hydrolyzed by pepsin.”’]

[Aside 3: The theme of this posting is the significance of Stanley’s feat of crystallizing tobacco mosaic virus. See Stanley’s 1946 Nobel lecture (1) for details on the heroic effort that went into that achievement.]

Importantly, and to the surprise of many, Stanley’s protein-containing TMV crystals retained the infectious activity of the actual virus! Also, it is crucially important that crystals are exquisitely pure. This key fact enabled Frederick Bawden and Norman Pirie in 1936 to demonstrate unequivocally that TMV is not a pure protein. Instead, TMV contains about 6% ribonucleic acid (RNA) (3). Consequently, whatever it is about TMV that enables it to produce copies of itself, that ability resides in its protein, or in its nucleic acid, or in a combination of its two macromolecular constituents (see Aside 4). [Aficionados might note that the ability of TMV to form crystals also implied that the virus has a regular structure.]

[Aside 4: Stanley may have initially believed that TMV is comprised entirely of protein. In his 1935 Science paper (2), he notes: “Although it is difficult, if not impossible, to obtain conclusive positive proof of the purity of a protein, there is strong evidence that the crystalline protein herein described is either pure or is a solid solution of proteins.”]

The finding that TMV is comprised of protein and RNA also gave rise to the notion that a virus is more complex than a mere chemical, even if not quite an organism. But note that Max Schlesinger in 1933 was actually the first one to find nucleic acid in a virus. Making use of new high-speed centrifuges, Schlesinger purified a bacteriophage to high purity and demonstrated that it is comprised of 50% protein and 50% DNA. However, Schlesinger did not study crystalline material, as Bawden and Pirie had done. Moreover, no one at the time knew quite what to make of Schlesinger’s findings. Consequently his work did not get as much attention as that of Bawden and Pirie.

Although Stanley’s work would eventually be recognized by the Nobel committee, when it first appeared many scientists could not accept that a crystal might actually possess a key property that we associate with life—the ability to replicate. And other researchers failed to see how an infectious mottling illness in tobacco plants could be relevant to disease in humans. [This point is reminiscent of the medical community’s disinterest in Peyton Rous’ 1911 discovery of a transmissible cancer in chickens. Medical researchers of the day could not see its relevance to malignancies in humans (4).]

Recall that many serious biologists and chemists in those earlier years still adhered to the belief that some “vital” force outside the known laws of chemistry and physics would be needed to explain the phenomenon of life. Yet if viruses are so simple that they that they could be crystallized like table salt, and still express that most fundamental property of living systems—the ability to replicate—then there might be reason to believe that the nature of biological replication indeed might be understandable in terms of conventional chemistry and physics. Moreover, note that crystallography is a very precise science. Thus, taken together, the facts that TMV could be crystallized, and yet retain biological activity, strongly implied to at least some scientists that conventional physics and chemistry would suffice to explain life.

Spurred on by this line of thought, a somewhat atypical group of investigators sought to understand the nature of genes. These researchers were atypical in that they generally had little or no knowledge of traditional genetics, or of biochemistry, or, in fact, of biology of any sorts. Many were physicists by background. But, they had a single goal in mind: to understand the physical basis of the gene. What’s more, several of these investigators recognized the advantages of focusing their research efforts on viruses.

This odd group’s interest in genes, and its focus on viruses, would lead to discoveries of singular overwhelming importance. Indeed, their research approaches and the results they generated gave rise to molecular biology. Thus, Stanley’s achievement would mark the death knell of vitalism and spur the beginning of the field of molecular biology (see Aside 5). And, when Watson and Crick solved the structure of DNA in 1953, it became clear that the expression and replication of the genetic material would be accounted for by the known laws of physics and chemistry.

[Aside 5: Physicist Max Delbruck was a key player in this atypical group of researchers, and he is recognized as one of the principal founders of the new science of molecular biology. Yet it is ironic that Delbrück was initially drawn to biology by the belief that it might reveal new concepts of physics. For more on Delbruck and the “phage group” he founded at CalTech, see reference 5.]

Wendell Stanley carried out his ground breaking research on TMV at the Rockefeller Institute (now the Rockefeller University). He passed away at a scientific conference in Salamanca, Spain in June, 1971.

References:

(1) Stanley, W.M., The isolation and properties of crystalline tobacco mosaic virus,
Nobel Lecture, December 12, 1946

(2) Stanley, W. 1935. Isolation of a crystalline protein possessing the properties of tobacco-mosaic virus. Science 81:644-645.

(3) Bawden F.C., N.W. Pirie, J.D. Bernal, and I. Fankuchen. 1936. Liquid crystalline substances from virus-infected plants. Nature 138: 1051–1055.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

Advertisements

Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob

I am intrigued by the genealogies of our leading scientists, since their mentors too were often preeminent scientists. Earlier postings noted the example of Jonas Salk, who did postgraduate studies under Thomas Francis; one of the great pioneers of medical virology, perhaps best known for developing the first influenza vaccine (1, 2). James Watson, who did his doctoral studies in Salvatore Luria’s laboratory, and Renato Dulbecco, who trained under both Luria and Max Delbruck (3), are other examples. In fact, Watson and Dulbecco shared a lab bench in Luria’s lab. Howard Temin did his doctoral (and postdoctoral studies too) in Dulbecco’s lab (4). And Delbruck, who hugely influenced the new science of molecular biology, did his doctoral studies under Max Born, the 1954 Nobel Laureate in physics. Moreover, Delbruck later served as an assistant to Lisa Meitner (5).

Important research paths were undertaken, and major contributions were made, which resulted from less formal interactions between budding young scientists and top scientists of the day. Howard Temin’s chance encounter with Harry Rubin, while on a mission to Dulbecco’s lab, is a case in point (4).

Our last posting told how Louis Pasteur came within a whisker of adding the discovery of viruses to his list of extraordinary achievements (6). Robert Koch played a part in that story for developing his famous postulates, which provided the standard for demonstrating that a particular microbe causes a particular disease.

The Pasteur article also noted that in 1898 Friedrich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. However, the piece did not point up that Loeffler had trained under Robert Koch. Also, it did not underscore the special significance of what Loeffler and Frosch achieved. In brief, by the 1890s Dmitry Ivanovsky and Martinus Beijerinck had independently discovered that the agent responsible for tobacco mosaic disease passes through bacterium-proof filters. Nevertheless, neither Ivanovsy nor Beijerinck appreciated the implication of their observation. Ivanovsky believed his filters might be defective, while Beijerinck thought the disease was caused by a “living liquid.” In contrast, Loeffler and Frosch, in addition to isolating the first virus that is pathogenic in animals, also carefully considered all possible explanations for their experimental findings, and then were the first to conclude the existence of a kind of microbe too small to be retained by bacterium-proof filters, and too small to be seen under a microscope, and that will not grow on laboratory culture media. They also correctly predicted that smallpox, cowpox, cattle plague, and measles are similarly caused by a “filterable virus.”

Loeffler made another major discovery, fourteen years earlier, in 1884, when he used his mentor’s postulates to identify the bacterium that causes diphtheria, Corynebacterium diphtheriae. Importantly, Loeffler also discovered that when he injected C. diphtheriae into animals, the microbe did not need to spread to the tissues it damaged. This observation led Loeffler to propose the bacteria were secreting a poison or toxin that spread to the remote sites and caused disease there.

Loeffler’s idea of a toxin was a new concept that subsequently was confirmed by Emile Roux, who had been Louis Pasteur’s assistant (6). Using bacterium-proof filters developed by Charles Chamberland in Pasteur’s lab, Roux showed that injecting animals with sterile filtrates of C. diphtheriae cultures caused death with a pathology characteristic of actual diphtheria. Roux was also a co-founder of the Pasteur Institute, where he was responsible for the production of diphtheria anti-toxin; the first effective diphtheria therapy. See Aside 1.

[Aside 1: Earlier, Roux suggested the approach Pasteur used to generate attenuated rabies virus for the Pasteur rabies vaccine (aging spinal cords from rabbits that succumbed to experimental rabies infections of their spinal cords). Roux later withdrew from the rabies project because of a disagreement with Pasteur over whether the rabies vaccine might be safe for use in humans (6).]

So, Loeffler and Roux trained under Koch and Pasteur, respectively. But why might toxin production by C. diphtheriae interest virologists. Well, in 1951, Victor Freeman at the University of Washington showed that the lethal toxins produced by C. diphtheriae (and by Clostridium botulinum as well) are the products of lysogenic bacteriophage carried by the bacteria. This was shown by the finding that avirulent strains of these bacteria became virulent when infected with phages that could be induced from virulent strains. So, are diphtheria and botulism due to bacteria or to viruses? Our chain of genealogies continues with a selective history of lysogeny.

Almost from the beginning of phage research (bacteriophage were discovered independently by Frederick Twort in Great Britain in 1915 and by Félix d’Hérelle in France in 1917), some seemingly normal bacterial cultures were observed to generate phage. Initially, this phenomenon was thought to be a sign of a smoldering, steady state kind of persistent phage infection. Then, during the 1920s and 1930s, the French bacteriologists, Eugene Wollman and his wife Elizabeth, working together on Bacillus megatherium at the Pasteur Institute, provided evidence that instead of a steady state infection, the phage actually enter into a latent form in their host cells; a form in which they might be harmlessly passed from one cell generation to the next. [Considering the state of knowledge back then, note the insightfulness of Eugene Wollman’s 1928 comment, “the two notions of heredity and infection which seemed so completely distinct and in some ways incompatible, . . . almost merge under certain conditions.”] See Aside 2.

[Aside 2: Since some bacterial strains would, on occasion, spontaneously undergo lysis and release bacteriophage, the cryptic bacteriophage they carried were called “lysogenic.” Thus, it is a bit odd that “lysogeny” eventually came to refer to the temperate relationship between these phages and their host cells.]

In the late 1930s, the Wollmans developed a close friendship with Andre Lwoff, their new colleague at the Pasteur Institute. The Wollmans introduced Lwoff to their ideas about lysogeny, but, as Lwoff confesses, he was not then impressed by bacteriophage (7).

The Nazi occupation of Paris during the Second World War began in 1940. From then on, the Jewish Wollmans were prevented from publishing their research findings. Nevertheless, they continued their research at the Pasteur Institute until 1943, when they were seized by the Nazis and sent to Auschwitz. They never were heard from again. Their friend, Lwoff, grieved their loss and became active in the French resistance, gathering intelligence for the Allies, while also hiding downed American airmen in his apartment.

After the war, Lwoff received several honors from the French government for his efforts against the Nazis. He also returned to his research at the Pasteur Institute, studying the genetics of Moraxella; a bacterial pathogen of the human respiratory tract. Because of his work as a microbial geneticist, he was invited to the 1946 Cold Spring Harbor Symposium, where he met Max Delbruck. And as happened to others, meeting Delbruck resulted in Lwoff being seduced by bacteriophage.

Andre Lwoff
Andre Lwoff

Back in Paris, Lwoff’s passionate interest in phages was heightened further by discussions with Jacques Monod, a friend of Max Delbruck, and Lwoff’s neighbor in the attic of the Pasteur Institute. Although Monod was Lwoff’s junior colleague (in fact, it was Lwoff who first stirred Monod’s interest in microbiology), Lwoff’s conversations with the future Nobel Laureate resulted in Lwoff becoming intensely fascinated by lysogeny, which he began to study in 1949 (7).

Because of Lwoff’s earlier friendship with the Wollmans, he chose to study a lysogenic strain of B. megatarium. And, making use of techniques he learned from Renato Dulbecco during a brief stint at Cal Tech, he was able to follow a single lysogenic bacterium, which enabled him to observe that a bacterium could go through multiple rounds of replication without liberating virus. What’s more, he discovered that the phages are released in a burst when the cell lyses, thereby dispelling the still current notion that phages are liberated continuously by lysogenic bacteria. Furthermore, Lwoff showed that lysogenic bacteria usually do not contain phage particles, since none are detected when the cells are experimentally lysed with lysozyme; confirming the earlier (1937) findings of the Wollmans.

Lwoff went on to show that temperate phage genomes are maintained in a previously unknown integrated state in their host cell, and he gave the integrated phage genomes a name, “prophage.” He also discovered, unexpectedly, that irradiating lysogenic bacteria with ultraviolet light could induce the temperate phages to emerge from their latent state, and then replicate in, and lyse their host cells. And, he discovered that the phages lyse their host bacterial cells by producing enzymes that destroy bacterial cell walls.

Prophage
Prophage

Lwoff’s elucidation of the fundamental nature of lysogeny in bacteria would later provide a paradigm for the DNA tumor viruses, the herpesviruses, the oncogenic retroviruses, and HIV. He was awarded a share of the 1965 Nobel Prize for physiology or medicine for his lysogeny research. He shared the award with his fellow Pasteur Institute scientists, François Jacob and Jacques Monod, who received their awards for their pioneering studies of gene regulation in E. Coli.

A rather intriguing aspect of this story is that Lwoff was joined in his research on lysogeny at the Pasteur Institute by Elie Wollman; the son of Eugene and Elizabeth. Elie, born in 1917, escaped from the Nazis in Paris in 1940 and worked in the French resistance as a physician. In 1946, after the war, he came to the Pasteur Institute, where he took its microbiology course and then became Lwoff’s research assistant. Then, in 1947, Elie too happened to meet Max Delbruck (in Paris in this instance) and was invited to join the Cal Tech phage group, where he spent the next two years. See Aside 3.

Elie Wollman
Elie Wollman

[Aside 3: By the early 1940s, the then young Cal Tech “phage group,” headed by Max Delbruck, was on its way to becoming the World’s great center for phage research (5). However, the American group had little interest in lysogeny, since Delbrück neither believed in it, nor saw its importance. Instead, Delbruck was totally committed to the study of lytic phages. Then, during the late 1940s, Delbruck began to lose interest in molecular biology and looked for new research directions. When he thought of turning his attention to brain function, he asked his group to put together a series of seminars based on papers written by prominent neuroscientists of the day. Elie Wollman was the only member of the Cal Tech group who declined to participate in that endeavor, since he was totally committed to bacteriophage. Moreover, Elie was the one who finally convinced Delbruck that “such a thing as lysogeny does exist (7).”

Elie himself tells us that when he looked into a bibliographical index at Cal Tech, he came across an index card referring to his parent’s 1937 paper, which reported their finding that lysogenic cells contain a non-infectious form of the phage (8). “Delbruck’s comment on the card was “Nonsense.”]

After Eli’s two-year stint with Delbruck in Pasadena, he returned to the Pasteur Institute. Meanwhile, Francois Jacob had come to the Institute in the hope of beginning a research career in genetics under the tutelage of either Lwoff or Monod. Before that, in 1940, Jacob, who also was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was later severely wounded at Normandy in August 1944, ending his dream of becoming a surgeon.

Francois Jacob
Francois Jacob

Initially, Jacob was spurned by both Lwoff and Monod, but was finally taken on by Lwoff, who suggested that he, Jacob, start work on “the induction of the prophage.” Jacob confesses he had no idea what that meant, but he accepted the project. Thus it came to pass that Francois Jacob and Elie Wollman established a particularly close and friendly collaboration, in which they turned their attention to the lambda prophage of E. coli. Their initial goal was to clarify the events of bacterial conjugation so that they might then understand the phenomenon whereby a temperate phage carried by a lysogenic bacterium is activated to undergo vegetative replication when that bacterium conjugates with, and transfers its integrated phage genome to a non-lysogenic bacterium.

To accomplish their goal, Wollman and Jacob began with experiments to locate the lambda genome on the chromosome of the lysogenic cell, and to follow its transfer during conjugation into a non-lysogenic recipient cell. A key feature of their experimental approach was conceived by Wollman (8). It was simply to interrupt conjugation between a lysogenic donor (Hfr) cell and a non-lysogenic recipient (F-minus) cell, at various times, by using a kitchen blender to break the mating cells apart. Using the blender to interrupt conjugation, and also using bacterial strains in which the recipient bacteria contained a set of mutations, and plating the mating mixture on selective media, Wollman and Jacob were able to measure the length of time required for each of the corresponding wild-type genes to be transferred from the Hfr donor cells to the F-minus recipient cells. Indeed, the time intervals between the appearances of each wild type gene in the recipient cells directly correlated with the distances between the genes, as independently determined by recombination frequencies. Thus, the interrupted mating approach gave Wollman and Jacob a new means to construct a genetic map of the bacterium, while also enabling them to locate the integrated phage genome on that map. Their experimental approach also allowed Wollman and Jacob to establish that, during conjugation, the donor cell’s genome is transferred linearly to the recipient cell. [The designation “Hfr” was coined by William Hayes because Hfr strains yielded a high frequency of recombinants when crossed with female strains.]

Importantly, Wollman and Jacob’s study of the activation of a lambda prophage when it enters a non-lysogenic F-minus recipient (a phenomenon they called “zygotic induction”), showed that the temperate state of the lambda prophage is maintained by some regulatory factor present in the cytoplasm of a lysogenic bacterium, but which is absent from a non-lysogenic one. It led to the discovery of a “genetic switch” that regulates the activation of the lysogenic bacteriophage, and of a phage-encoded repressor that controls the switch. These findings are among the first examples of gene regulation, and are credited with generating concepts such as the repressor/operator, which were firmed up by Jacob and Monod in their Nobel Prize-winning studies of the E. coli lac operon. See Aside 4.

[Aside 4: At the time of Wollman and Jacob’s interrupted mating experiments, kitchen blenders had not yet made their way to European stores. Eli was aware of these appliances only because of his earlier stint at Cal Tech. He bought a blender for his wife before returning to France, and then “borrowed” it for these experiments.]

Wollman and Jacob went on to demonstrate that the fertility or F factor, which confers maleness on the donor bacteria, can exist either in an integrated or an autonomous state. Indeed, this was the first description of such a genetic element, for which they coined the term “episome;” a term now largely replaced by “plasmid.”

Wollman and Jacob also determined that the E. coli chromosome is actually a closed circle. The background was as follows. Only one F factor is integrated into the chromosome of each Hfr strain, and that integration occurs at random. And, since the integrated F factor is the origin of the gene transfer process from the Hfr cell to the F-minus cell, interrupted mating experiments with different Hfr strains gave rise to maps with different times of entry for each gene. However, when these time-of-entry maps were taken together, their overlapping regions gave rise to a consistent circular map. The discovery of the circular E. coli chromosome was most intriguing, because all previously known genetic maps were linear. See Aside 5.

[Aside 5: The bacterial strain used by Wollman and Jacob in their study of zygotic induction was, in fact, the original laboratory strain of E. coli (i.e. E. coli K12) that was isolated in1922 from a patient with an intestinal disorder. In 1951, Esther Lederberg discovered that K12 is lysogenic. The discovery happened when she accidentally isolated non-lysogenic or “cured” derivatives of E. coli K12 that could be infected by samples of culture fluid from the parental K12 strain, which sporadically produced low levels of phage. Esther gave the lysogenic phage its name, lambda.

Esther was the wife of Joshua Lederberg, who received a Nobel Prize in 1958 for discovering sexual conjugation in bacteria, and the genetic recombination that might then ensue. Prior to Lederberg’s discoveries, genetic exchange and recombination were not believed to occur in bacteria. Lederberg’s Nobel award was shared with George Beadle and Edward Tatum (the latter was Lederberg’s postdoctoral mentor) for their work in genetics.

Joshua Lederberg, working with Norton Zinder (9), also discovered transduction, whereby a bacterial gene can be transferred from one bacterium to another by means of a bacteriophage vector. And, working together with Esther, Joshua discovered specialized transduction, whereby lambda phage transduces only those bacterial gene sequences in the vicinity of its integration site on its host chromosome. Esther and Joshua also worked together to develop the technique of replica plating, which enabled the selection of bacterial mutants from among hundreds of bacterial colonies on a plate and, more importantly perhaps, to provide direct proof of the spontaneous origin of mutants that have a selective advantage.]

In 1954 Elie Wollman was appointed a laboratory head in his own right at the Pasteur Institute. He retired from research in 1966 to become vice-director of the Institute, which he then rescued from a severe financial crisis. He continued to serve in that role for the next 20 years, while garnering numerous prestigious awards for his research and service.

Francois Jacob earned his doctorate in 1954 for his lysogeny studies. Then, realizing that he and Jacques Monod, his senior neighbor in the Pasteur Institute attic, were actually studying the same phenomenon, gene repression, he entered into a hugely productive collaboration with Monod that led to the elucidation of the genetic switch that regulates beta-galactosidase synthesis in E. coli (9). Their collaboration established the concepts of regulator genes, operons, and messenger RNA, for which they shared in the 1965 Nobel Prize for physiology or medicine, as noted above. See Asides 6 and 7.

Jacques Monod
Jacques Monod

[Aside 6: One of Jacob and Monod’s first experiments was the famous 1957 PaJaMa experiment, carried out in collaboration with Arthur Pardee, who was then on sabbatical at the Pasteur Institute. In brief (for aficionados), a Lac-positive, Hfr strain was grown in an inducer-free media, and then mated, still in an inducer-free media, with a Lac-minus, F-minus strain. (Note that the deletion in the Lac-minus, F-minus strain included the LacI gene, which encodes the yet to be discovered lac repressor.) As expected, in the absence of inducer, no beta-galactosidase is detected initially. But, after the donor DNA sequence, which bears the normal Lac genes (including LacI), is transferred to the Lac-minus recipient, it initially finds no repressor in the recipient cell and begins to synthesize beta-galactosidase. Then, as the donor cell’s lac repressor gene begins to be expressed in the recipient cell, in the inducer-free media, expression of the donor cell’s beta-galactosidase gene ceases. The PaJaMa experiment thus showed that the genetic regulation of enzymatic induction depends on a previously unknown regulatory molecule, the repressor.

Notice the similarity between the rationale for the PaJaMa experiment and that of the earlier Wollman and Jacob experiment on zygotic induction. In each instance, a process regulated by a repressor is suddenly in the repressor-free environment of a recipient cell.]

[Aside 7: In June of 1960, Francois Jacob, Matt Meslson, and Sidney Brenner came together in Max Delbruck’s Cal Tech lab to carry out an experiment that confirmed the existence of messenger RNA. The key to the experiment was their ability to distinguish ribosomes present in the cell before infection from ribosomes that might have been made after infection. They cleverly did that by incorporating heavy isotopes into ribosomes before infection, so that they might be separated in a density gradient from ribosome made after infection. Then, they showed that RNA produced by T2 phage in E. Coli associates with ribosomes that were synthesized by the cell entirely before infection. Furthermore, the new phage-specific RNA directs the synthesis of phage-specific proteins on those “old” ribosomes. I vote for this experiment as the most elegant in the entire history of molecular biology (11).]

Incidentally, during the Nazi occupation of Paris, Monod too was active in the French Resistance, eventually becoming chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service.See Aside 7.

[Aside 7: I am singularly intrigued by the experiences of Andre Lwoff, Elie Wollman, Francois Jacob, and Jacques Monod during the Second World War. References 3 and 5 recount the wartime experiences of Renato Dulbecco and of Max Delbruck, and of other great scientists of the time. Other posts on the blog give accounts of virologists courageously placing themselves in harm’s way under different circumstances. Examples include pieces featuring Ciro de Quadros, Carlo Urbani, Peter Piot, and Walter Reed.]

References:

(1) Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posted on the blog March 27, 2014.

(2) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 25, 2014.

(3) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog, December 4, 2013.

(4) Howard Temin: “In from the Cold,” Posted on the blog December 16, 2013
(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.

(6) Louis Pasteur: One Step Away from Discovering Viruses, Posted on the blog January 7, 2015.

(7) Lwoff, Andre, The Prophage and I, pp. 88-99, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(8) Wollman, Elie L, Bacterial Conjugation, pp. 216-225, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J.D. Watson eds., Cold Spring Harbor Laboratory Press, 1966.

(9) “The Phage in the Letter,” Posted on the blog November 4, 2013.

(9) Francois Jacob, Nobel Lecture, December 11, 1965.

(11) Norkin, Leonard C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010.

Andre Lwoff

Seymour Benzer: A Star of the “Golden Age of Molecular Biology”

An earlier blog posting told of how Max Delbruck, in 1950, summoned Renato Dulbecco to his office to propose that Dulbecco launch animal virus research at Caltech; where virology was still concerned solely with bacteriophages (1.) The background was as follows.

In the late 1940s, a wealthy Californian became ill with shingles (later known to be a delayed complication of chickenpox, caused by varicella-zoster virus, a herpesvirus). The man’s physician explained that nothing could be done for his shingles, and moreover, that virtually nothing was known about the viruses that infect humans. Auspiciously, the physician knew of the studies being done on bacteriophages at Caltech, and he also was aware that Caltech was the great center for such work. So, after explaining to his well-heeled patient that bacteriophages were only of theoretical interest regarding human disease, he suggested that the patient might help to develop a center at Caltech which might begin to study medically important viruses. The patient agreed, and since virology at Caltech was headed by Delbruck, the former physicist found himself with an endowment to study human viruses, with virtually no background for how to use it.

Delbruck tried to recruit Dulbecco to open up animal virus research at Caltech because Dulbecco, unlike others in the Caltech “Phage Group,” trained as a physician. However, Dulbecco was not the only one who Delbruck sought to enlist to take up the task that day. As Dulbecco tells us (2), “One day Seymour Benzer and I were called to his (Delbruck’s) office: Delbruck pointed out that animal virology appeared ready for major advances. Would either of us be interested in trying his hand at it? To me it sounded wonderful. I had been thinking perhaps with nostalgia, of my work with tissue cultures, years before, in Guiseppe Levi’s laboratory in Torino; so I immediately expressed my interest, before Benzer could say anything. Benzer, on the other hand, was not interested, so everything was settled without delay.”

Thus, it came to pass that Dulbecco was the one who launched the study of animal virology at Caltech and, moreover, the one who initiated quantitative animal virology in general. But, who was Benzer, and what became of him?

As an undergraduate at Brooklyn College in the late 1930s, Seymour Benzer considered majoring in biology. However, since the biology teaching of the day was largely concerned with taxonomy, which he had little interest in, he instead majored in physics. He continued his training in physics as a graduate student at Purdue University during the Second World War.

His doctoral research involved semi-conductors, as part of a project to develop better crystal rectifiers; a crucial component for radar. Benzer’s doctoral work as a physicist is notable since it is credited with contributing to the development of the first transistors.

Apropos the current story, while Benzer was studying physics at Purdue, he happened to read Erwin Schrodinger’s 1944 book: What is Life? A chapter in Schrodinger’s book, entitled “Delbruck’s Model,” especially intrigued Benzer; so much so that he considered switching back to biology.

[Aside 1: Schrodinger, the great Austrian physicist and Nobel laureate, was at the time an anti-Nazi émigré, living in Ireland. In that regard, see references 1 and 5.]

Why might What is Life? have made such a strong an impression on Benzer? It was largely because it was a time when the chemical nature of the genetic material, and its manner of replication and action, were not yet known. In fact, most biologists thought that proteins constitute the genetic material, while DNA was merely a structurally uninteresting, monotonous molecule, much like a starch.

[Aside 2: The classic 1952 blender experiment of Alfred Hershey and Martha Chase, together with the earlier (1944) transformation experiments of Avery, MacLeod, and McCarty, would eventually convince virtually everyone that DNA is the genetic material. Additionally, the 1953 discovery of the DNA structure by Watson and Crick, would immediately suggest a plausible mechanism by which DNA might be replicated.]

Given the state of knowledge in the mid 1940s, when genes were still thought to be comprised of protein, the models of the day to account for how genes might be replicated and expressed were neither convincing nor satisfying. Consequently, many scientists came to believe that it would be impossible to understand heredity and gene function in terms of the known laws of chemistry and physics.

In What is Life? Schrodinger sought to account for genetic attributes in terms of quantum mechanics. For instance, to explain how genes might preserve their structure, and store genetic information over the lifetime of an organism, while at 310 degrees above absolute zero, he suggested that genes might reside in an aperiodic crystal state, in which their atoms stay put in stable energy wells. The Delbruck model that Schrodinger cites, which so excited Benzer, “explains” gene mutations as different quantum mechanical energy levels of a gene (3). [The Delbruck model may actually have inspired Schrodinger to write What is Life?]

Bearing in mind that Schrodinger was a Nobel laureate, who discovered the immensely important wave equation (which expressed the movements of electrons in terms of wave mechanics rather than as particles), we can appreciate the impact that his following comment (in What is Life?) may have had on some physicists of the day: “From Delbruck’s general picture of the hereditary substance, it emerges that living matter, while not eluding the ‘laws of physics’ as established up to date, is likely to involve ‘other laws of physics’ hitherto unknown which, however, once they have been revealed will form just as integral part of this science as the former.”

The notion, that “other laws of physics” might be discovered by researching the genetic material, roused Benzer to enter, and indeed help to create the field now known as molecular biology (4, 5).

Seymour Benzer (right), with Francis Crick in 1964
Seymour Benzer (right), with Francis Crick in 1964

[Aside 3: Surprisingly, Schrodinger himself seemed unaware of the earlier pioneering work of George Beadle, Boris Ephrussi, and Edward Tatum in the 1930s and early 1940s, which established the concept, “one gene, one enzyme;” later revised to “one gene, one polypeptide chain.” [Those ground-breaking biochemical genetic studies were carried out using the fungus Neurospora crassa.] Also, it is surprising that Schrodinger appears unaware that in 1940, Delbruck, together with Salvatore Luria and, eventually, Alfred Hershey, had already formed the “Phage Group,” which carried out its first experiments at the Cold Spring Harbor Laboratory on Long Island, NY, with the ultimate purpose of understanding the physical basis of heredity (4, 5).]

[Aside 4: James Watson refers in the following comment to an early time in his graduate student years at Indiana, while he was still deciding whose lab to join there: “Some weeks later in Luria’s flat, I first saw Max Delbruck, who had briefly stopped over in Bloomington for a day. His visit exited me, for the prominent role of his ideas in What is Life? made him a legendary figure in my mind. My decision to work with Luria had, in fact, been made so quickly because I knew that he and Delbruck had done phage experiments together and were close friends (6).]

Here now is one of my favorite parts of this story. Benzer, now leaning towards biology, was attending a meeting of the American Physical Society in Bloomington, Indiana, where he happened to accompany a friend to the home of the friend’s former classmate, who just happened to be the wife of Salvatore Luria. Benzer tells us, “I could not have been more impressed…and it was not long before he (Luria) had persuaded me to enroll in the phage course at Cold Spring Harbor. Thus I suddenly plunged into the biology business (6).”

[Aside 5: Incidentally, in 1936, Dulbecco was in Luria’s lab in Italy, while studying for his medical degree at the University of Torino. Having favorably impressed Luria, Dulbecco was later (after the Second World War and a brief stint in politics) invited to join Luria’s group at Indiana to study bacteriophages. Dulbecco and Watson shared a lab bench in Luria’s Indiana lab.]

Benzer next spent a postdoctoral year at the Oak Ridge Biology Division, and then had the choice of going to Salvador Luria’s laboratory at Indiana, or to Delbruck’s group at Caltech. Benzer relates, “…I asked Luria’s student James Watson for advice…Luria, he said, would be likely to ask me every day what I had done, whereas I might not see Delbruck for a week at a time. I chose to join Delbruck at Cal Tech (7).”

Benzer’s key contributions to the developing field of molecular biology took place mainly at Purdue, to which he returned after spending two years as a postdoctoral fellow in Delbruck’s Caltech lab. But first, here is a brief personal recollection. When I initially encountered genetics in high school in the 1950s, chromosomes were depicted as beads on a string, with the beads representing the genes. The beads (genes) were the units of function, determining whether you had blue or brown eyes, for example. An entire bead (each one representing a gene) was also the unit of mutation. Moreover, recombination occurred between the beads. Thus, each bead (gene) was the unit of function, mutation, and recombination.

By the late 1950s, it was reasonable to believe that a phage genome might well be one long thread of DNA. With that premise in mind, Benzer proposed that there might then be a uniform probability of recombination anywhere along the length of the phage genome. Note here the corollary notion that the unit of genetic function and the units of recombination, and perhaps mutation as well, are not necessarily the same physical entities.

Benzer carried out his experiments using T4 phage, specifically investigating the rII region of the T4 genome. Mutations in the T4 rII region cause infected cells to undergo premature (rapid) lysis, resulting in lower phage yields. The r (rapid lysis) mutants could be distinguished from wild-type T4 by their plaque morphology on E. coli strain B. Fortuitously, r mutants can not grow on E. coli strain K. Thus, T4 r mutants could be plaque-isolated on E. coli B and, if recombinants were to occur between r mutants, they might be detected on E. coli K.

When Benzer became aware of these facts, he realized that he had the ingredients at hand for a high resolution genetic system that might enable him to detect recombinants between mutations within the rII region; possibly even between mutations within the same gene. And, if one were to “run the genetic map into the ground” (as Delbruck put it), it might be possible to obtain recombination even between adjacent nucleotides.

So, Benzer infected E. coli K cells with pairs of independently isolated T4 rII mutants. And, as he hoped, he found that wild-type T4 recombinants indeed were generated, although at a very low frequency, which indicated that the rII mutations are very close together on the phage chromosome. But, and importantly, in addition to finding rare genetic recombinants between rII mutations, Benzer also found that certain pairs of rII mutants actually replicated together in E. coli K. That is, they complemented each other.

Next, Benzer found that the rII mutants could be placed in either of two groups, designated A and B. All A mutants complemented all B mutants, and visa versa. However, mutants within the same group could not complement each other. Moreover, for complementation to occur, the mutations also had to be on separate phage chromosomes; that is, they had to be in trans. Complementation did not occur if the mutations were on the same phage chromosome; that is, in cis. [In the trans orientation, one phage chromosome contains a wild-type rIIA region and a mutant rIIB region, while the other phage chromosome contains a mutant rIIA region and wild-type rIIB region. In the cis orientation, both mutations are on the same phage chromosome, and no wild-type RII regions are present.]

Thus, in addition to demonstrating that all of the rII mutations are very close together on the T4 chromosome, Benzer’s experimental results also showed that the rII mutations fall into two distinct complementation groups. The key question is the explanation for complementation between rIIA and rIIB mutants, but only when the mutations are expressed in trans. The answer is that the rIIA and rIIB regions of the phage chromosome are separate genetic units of function, each of which encodes a distinct polypeptide. Thus, if the rIIA and rIIB mutations are on separate phage chromosomes (i.e., in trans), then a wild-type A and a wild-type B polypeptide can be generated by the respective wild-type rII region of each chromosome, thereby enabling complementation.

Benzer dubbed the genetic units of function, as exemplified by the rIIA and rIIB regions, “cistrons,” since they are operationally defined by the cis-trans test (i.e., mutations in separate cistrons complement each other when expressed in trans, but not when expressed in cis). As expected, mutations in the same complementation group also cluster together on the phage chromosome, as shown by genetic mapping techniques.

To appreciate the immense significance of Benzer’s findings from his rII system, we need to remember that classical genetics made no distinction between genes as units that specified a particular phenotypic trait, versus units of mutation, or as units of recombination. Indeed, classical genetics envisioned a gene as a single indivisible unit that embodied all three of these properties. Benzer’s experiments thus provided the distinctions between genetic units of function (cistrons), versus units of recombination, and of mutation, making clear that a gene is a unit that encodes a polypeptide, whereas a single nucleotide is the minimal unit of mutation. And, recombination might occur even between single pairs of nucleotide bases.

Benzer’s cis-trans test was widely used to determine whether any two mutations are in the same or different functional genetic units. [Notice that the the cis-trans test reflects the earlier one gene-one protein (now one gene-one polypeptide chain) concept.] Today, the term “cistron” is rarely used. Instead, we simply say gene to imply the same meaning.

Benzer also examined a curious rII mutation, r1589, which contains a deletion that extends over portions of both the A and B cistrons, including the spacer region between them. This mutation leads to the production of a continuous polypeptide chain comprised of portions encoded by both the A and B cistrons. The study of r1589 led to important insights into how mRNA (yet to be discovered) is transcribed and then translated into protein.

By the 1960s, Benzer’s interest in genetic fine structure began to wane. Yet he was still publishing papers at a steady rate. The simultaneous appearance of several of his papers tempted Delbruck to append the following postscript to a letter from his wife to Benzer’s wife: “Dear Dotty, please tell Seymour to stop writing so many papers. If I gave them the attention his papers used to deserve, they would take all my time. If he must continue, tell him to do what Ernst Mayr asked his mother to do in her long daily letters, namely underline what is important (8).”

Benzer’s reaction was: “It is very difficult for me now to think of anything worthy of being underlined.” So, Benzer’s scientific focus shifted again; this time to developing a model system that might lead to insights into the genetic basis for behavior. He eventually settled on using Drosophila melanogaster, and founded the field of neurogenetics.

Seymour Benzer passed away in November, 2008. He received numerous awards for his research, including the National Medal of Science, but not the Nobel Prize, which many believed he deserved.

References

(1) Renato Dulbecco and the Beginnings of Quantitative Animal Virology, Posted on the blog December 4, 2013.

(2)  Dulbecco, Renato, The Plaque Technique and the development of Quantitative Animal Virology, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(3)  Stent, Gunther S., Introduction: Waiting for the Paradox, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(4) Norkin, Leonard C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010. Chapters 1 and 2 recount the beginnings, philosophy, and early contributions of the Phage Group.

(5) Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013. This piece contains more background on Max Delbruck, Salvatore Luria, and the founding of the phage group, as well as some of my very favorite anecdotes.

(6) Watson, J. D., Growing up in the Phage Group, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(7) Benzer, Seymour,  Adventures in the rII region, in Phage and the Origins of Molecular Biology, J. Cairns, G.S. Stent, and J. D. Watson eds., Cold Spring Harbor Laboratory of Quantitative Biology, 1966.

(8) Sidney Brenner: “Only Joking”, Posted on the blog January 7, 2014. This piece gives another glimpse into the personality of Max Delbruck.