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

[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.

The American Public’s Response to the 2014 West African Ebola Outbreak: Update

A premise of my August 10, 2014 blog entry was that public fear of Ebola in the United States has been disproportionate to the actual threat that the virus poses here (1). The piece also discussed the factors that shape the public’s reaction to particular viral diseases. The viruses discussed were Ebola, influenza, polio, and HIV.

For a sense of my earlier argument, consider that influenza kills on average about 40,000 Americans (and 500,000 people world wide) yearly. In contrast, at the time of the August 10 blog entry, the current Ebola outbreak was estimated to have killed about 1,000 people in total. Moreover, the largest previous Ebola outbreak, which occurred in Uganda in 2000, claimed 244 lives. Furthermore, up to the time of the earlier posting, Ebola had killed a total of about 2,000 people since it first emerged in 1976. All Ebola outbreaks occurred in Africa, and no Ebola infection had ever occurred in the United States. In each of the previous Ebola outbreaks, the virus ran its destructive course and then “disappeared.” Yet the American public is far more troubled by Ebola than it is by influenza. Indeed, an influenza vaccine is available to prevent influenza, yet all too few individuals avail themselves of it. And, while no one has yet contracted Ebola in the United States, the public has been clamoring for an Ebola vaccine.

Subsequent to my earlier blog entry, several key developments have taken place, one of which has been receiving virtually minute-by-minute cable news coverage. As most everyone knows, Thomas Duncan, a Liberian, arrived at the Dallas/Fort Worth International Airport on September 20; a day after he boarded an airplane in Monrovia, Liberia. Ten days later, Duncan was diagnosed with Ebola at the Texas Health Presbyterian Hospital in Dallas. Mr. Duncan was the very first person in the current outbreak to be diagnosed with Ebola outside of Africa, and he was the very first person to develop symptoms in the United States.

A second key development is that the Centers for Disease Control and Prevention (CDC) now predicts that the current outbreak in West Africa might be vastly more devastating than any previous Ebola outbreak. The CDC reported on September 23 that, in a worst-case scenario, African Ebola cases could reach 1.4 million in four months time. [It is not entirely clear why the current outbreak has been so devastating. But, the earlier Ebola outbreaks happened in rural African villages, where infected individuals had fewer contacts. The current outbreak has spread to more densely populated urban areas, where infected individuals might come into contact with more people over a wider area.]

How might these new developments affect the premise of the earlier piece? Read on.

We begin by considering the implications of Mr. Duncan entering the United States, while carrying the Ebola virus. First, in the modern era of world-wide jet travel, a virus outbreak could spread across the globe in a day’s time (2). Second, airport screening is inherently porous. Moreover, it may be particularly so in the case of Ebola since the incubation period for symptoms to emerge can be as long as 21 days. Thus, an Ebola-infected individual, who is symptom-free, might board an airplane and later disembark anywhere in the world, and afterwards develop symptoms. In fact, Mr. Duncan was screened for fever at the airport in Monrovia; a standard practice there. His temperature was normal, and he was allowed to board his flight out of Liberia.

Airport screening must also rely on travelers being aware of their exposure to the virus, and on their integrity to report their exposure. In this regard, Duncan also stated on an airport form in Monrovia that he had not been exposed to Ebola. It is not clear whether Duncan knew that he had been exposed. Regardless, Liberian officials announced plans to prosecute him when he returns to Liberia, and Texas prosecutors too are considering bringing charges against him. [Duncan was exposed on September 15 in Monrovia, while playing the part of a Good Samaritan. He helped a pregnant woman, stricken by Ebola, get to a hospital. But, the woman was turned away by the hospital because of lack of space in its Ebola ward. She was taken back home and died later that evening. Duncan’s condition in Dallas has been critical for the past several days.]

Some in Congress, and others on the campaign trail, have been calling for banning passengers arriving from West Africa. However, federal officials have rejected that notion. Importantly, such a step would prevent medical workers and other assistance from reaching Africa. And it is crucially important that this not happen. Aside from ethical and humane considerations, the way to finally end the threat of Ebola in the United States is to stop the epidemic at its source (see below).

Considering that it was only a matter of time before Ebola-infected individuals might pass through screening procedure and arrive in the United States, the CDC and hospitals and health departments around the country have been preparing for that event. And, since people with Ebola are not contagious until symptoms develop, and contracting the virus requires contact with a sick person’s bodily fluids (e.g., blood or vomit), an Ebola outbreak should be quickly contained here by carrying out basic public health procedures (i.e., isolating infected individuals, and tracing and isolating their contacts).

That said, there is much that is troubling about the response of local and federal health officials in the incident involving Mr. Duncan in Dallas. The first time that Duncan came to the emergency room at Texas Health Presbyterian Hospital, he told a nurse that he had just been in Liberia. [Liberia, Guinea, and Sierra Leone are the three West African countries where Ebola is rampant]. However, this information was not transmitted to the doctors who treated Duncan. Believing that Duncan had a low-grade fever from a viral infection, they sent him home with antibiotics (hmm?). The hospital later released a statement blaming a flaw in its electronic health records system for its decision to send Duncan home. There were separate “workflows” for doctors and nurses in the records, so that doctors were not aware Duncan had come from Africa.

Three days after Duncan was sent home from the Dallas hospital, he came back with worsening symptoms. This time, he was placed in isolation, and both the CDC and a state lab in Texas then confirmed his condition as Ebola. Alarmingly, 114 people, including several schoolchildren, were believed to have been in contact with Duncan during the few days between his first visit to the hospital and his return and isolation there.

The number of people being monitored in Dallas has since dropped to ten, and none of these individuals has shown signs of infection. Regardless, public anxiety mounted after Duncan’s diagnosis was announced and his contacts were quarantined. Reports of worried Dallas parents keeping their children home from school were reminiscent of the public’s response to the polio outbreaks of the 1950s (1).

Astonishingly, the four people, who Duncan had been living with until his hospitalization, were forced to remain in the apartment they had been sharing. The apartment had not been sanitized, and the sheets and towels that Duncan had been using remained there. The explanation offered by the Texas health commissioner was that officials had encountered “a little bit of hesitancy” in finding a contractor willing to clean the apartment. And, while county officials visited the apartment, they did so without wearing protective gear. The four people Duncan was living with have since been moved from the potentially contaminated apartment.

Important lessons should have been learned from the missteps in Dallas. And, despite those missteps, no one in Dallas appears to have contracted Mr. Duncan’s infection. Thus, we might remain optimistic about the ability of our public health system to contain an Ebola outbreak anywhere in the United States. Moreover, that optimism might be bolstered by the example of Nigeria, the most populous nation in Africa (177,000,000 individuals), which recently contained its first Ebola outbreak. Interestingly, Nigeria’s outbreak grew from a single airport case, and it was contained using basic public health procedures. Nigerian health workers made nearly 18,500 face-to-face visits to monitor the nearly 900 people who had contact with known cases. Incidentally, the Bill & Melinda Gates Foundation financed the creation of the Ebola Emergency Operations Center, which oversaw the Nigerian response. With the best public health infrastructure in the world, we ought to be able to do as well here.

Although the epidemic in Nigeria was contained, the epidemic rages out of control only a few hundred miles away, in the epicenter comprised of Liberia, Sierra Leone, and Guinea. These underdeveloped and poverty-stricken nations have altogether inadequate public health systems that are overwhelmed by the scale of their epidemics. Thus, the Nigerian experience is not applicable in those countries, which desperately need massive international assistance.

President Obama announced on September 16 that the United States would send about 3,000 American military personnel, including doctors, to Liberia and Sierra Leone, to help construct Ebola treatment centers there, and to train up to 500 health-care workers per week. Importantly, and as noted above, irrespective of ethical and humane considerations, the epidemic must be stopped at its West African source, before we might be entirely safe from it here. But that will not be an easy or quickly realized task, since current estimates are that it will require as many as 30,000 health-care workers to bring the West African epidemic under control. The international community will need to become engaged in that effort to a vastly greater extent than it has to date.

In the interim, considering that Ebola does not spread nearly as easily as the viruses in doomsday movies do (recall that Ebola can be contracted only by contact with the bodily fluids from a person who has developed Ebola symptoms), and considering the asserted excellence of the public health infrastructure in the United States, Ebola still poses less of a threat here than influenza does, and less of a threat than several other viruses as well. [See the Postscript, below.]

References

(1) The American Public’s Response to the 2014 West African Ebola Outbreak, posted on the blog August 10, 2014.

(2) Carlo Urbani: A 21st Century Hero and Martyr, posted on the blog February 11, 2014.

Related

Peter Piot: The Discovery of Ebola Virus, posted on the blog September 2, 2014.

Postscript

As long as the epidemic lasts, new developments are bound to happen that cause us to reevaluate our earlier perspective. Just yesterday, a nurse in Madrid became the first health worker known to contract Ebola outside of West Africa. She was infected while attending to a Spanish missionary who contracted the illness in Sierra Leone. The missionary was flown to the Carlos III hospital in Madrid, where he succumbed three days afterward. The nurse was in his room in the Madrid hospital only twice; once before his death, and once afterwards, and she was wearing protective gear. This incident, together with the episode in Dallas, causes us to question just how well prepared Western health care systems actually are to safely treat people with Ebola, while not endangering their health workers or the public.

Addendum: October 8, 2014

Thomas Duncan passed away today at the Texas Health Presbyterian Hospital in Dallas.

Also, an editorial in Nature this week (9 October 2014) makes several points that supplement the above discussion. In particular, “even in rich countries, inequalities in access to health care and cost-cutting in the health services can create vulnerabilities… Were Ebola to spread in underprivileged urban areas, it might not be so easy to control as US officials are making out. The uninsured, in particular, may think twice about going to see a doctor, and so hamper efforts to stem an outbreak.”

And, regarding the disproportionate and excessive coverage by the America media of Ebola here in the USA, “People who suspect they might have been in contact with someone infected with Ebola might now be reluctant to come forward in case their names are splashed all over the headlines. The public has a legitimate interest in knowing the places an infected person has frequented, for example, but there is a fine line between this and blatant voyeurism, invasion of privacy and sensationalism.”