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?
Seymour Benzer majored in biology as an undergraduate at Brooklyn College in the late 1930s, but he was a graduate student in physics 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).
[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.
(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.