An Interview with Manuel F. Varela and Ann F. Varela: Who is Mark Ptashne? And What is Genetic Switching and Phage Lambda Concerns?

Dec 14, 2020 by

Mark Ptashne

He pushes and pushes on a single tiny point absolutely relentlessly.

–Dr. Alexander Gann

Everything we know about gene transcription seems to have come out of Mark Ptashne’s lab. Or was done elsewhere by people who had been in Mark Ptashne’s lab. Or was done elsewhere by people who were inspired by Mark Ptashne’s ideas.

–Dr. Guido Guidotti

Ptashne’s successful search for, and characterization of, the elusive repressor of bacteriophage λ, work that spanned two decades, can fairly be regarded as the greatest sustained experiment of the last century.”

–Joseph Sambrook

A few simple principles underlie gene regulation across the board, from bacteria to yeast, plants, fruit flies, and humans.

–Dr. Mark Stephen Ptashne

Michael. Shaughnessy

1) Born in the windy city of Chicago, apparently Mark Ptashne did not stay there for long—perhaps due to the cold—but for the record, when exactly was he born, and where did he do his early educational studies?

Dr. Mark Ptashne is world-renowned for his discoveries on a molecular mechanism involving gene regulation. One of his most noteworthy scientific contributions revolves around the purification of the so-called phage lambda (λ) repressor protein and elucidating how it functions to regulate genes.

Mark Stephen Ptashne was born in Chicago, Illinois, on the fifth of June in 1940. When Ptashne’s father Fred went into business with his brother Nate, the family moved to Minneapolis, Minnesota. They specialized in snowsuits and then candy in a family business. His mother, Mildred (Millie), was a social worker turned travel agent.

Ptashne graduated from Reed College in Portland, Oregon, in 1961 with a B.A. in Chemistry. Ptashne was a Junior Fellow of the Harvard Society of Fellows for three years. He took his Ph.D. in Molecular Biology from Harvard University in 1968.

2) Ptashne studied for his Ph.D. at Harvard—then apparently, he began teaching there—what was he working on as he must have impressed someone there?

In 1968, Dr. Ptashne would describe a “genetic switch” that turned a lambda (λ) virus on and off. The switch mechanism helped bacteriophages convert bacterial infection back and forth between the lytic and lysogenic modes.

Dr. Walter Gilbert and his colleague, Benno Müller-Hill at Harvard, described a switch that turned the cell’s ability to digest lactose on and off during metabolism. Ptashne joined the faculty at Harvard University as a Lecturer of Biochemistry and Molecular Biology in 1968. After his successful completion of his Ph.D. In 1971, Ptashne became a Professor there. He studied how proteins bind DNA and activate or repress transcription, beginning with phage λ and then turning to eukaryotes, predominantly yeast.

As an undergraduate at Reed College, Ptashne became fascinated with the work of Jacques Monod and François Jacob. They had discovered the celebrated lac operon. The lives and classic studies of Jacob and Monod are featured in Chapter 26 from our hardback “The Inventions and Discoveries of the World’s Most Famous Scientists.”

Back in the fall of 1960, when 26-year-old Ptashne arrived at Harvard, he brought a leather jacket, golf clubs, a violin, and advice from Franklin Stahl. The latter was at Oregon, where Ptashne did summer research. Stahl told Ptashne that at Harvard, he should study under Professor Matthew Meselson (see Chapter 14). Ptashne had expressed interest in purifying the “Holy Grail” of genetic regulation, the λ repressor. However, Meselson and James Watson, both of whom were Harvard faculty, advised Ptashne that the isolation of a protein was risky. The consequences were dire if he failed. Ptashne would have no basis for a Ph.D. thesis if he could not invent a way to purify the λ repressor.

Instead, Ptashne was relegated to undertaking a “boring” but more reliable Ph.D. thesis project. He settled on conducting a routine genetic analysis of the bacteriophage λ. First, he found that bacteriophage λ DNA attached to the Escherichia coli genomic DNA. Next, Ptashne studied the transfer of phage λ DNA from a donor Escherichia coli cell to a recipient cell. He demonstrated that the newly acquired phage λ DNA underwent replication. Ptashne published these two relatively minor research projects in 1965 in the Journal of Molecular Biology.

With his Ph.D. thesis work now primarily completed in the summer of 1965, Ptashne was granted a three-year appointment by Harvard’s Society of Fellows program. He was now free to undertake the riskier task of finding the λ repressor. At first, Ptashne planned to invoke the new RNA-DNA hybridization technique to see the λ repressor, but the experiments failed to work effectively.

Next, Ptashne tried an approach where he subjected bacteria infected with bacteriophage λ to massive amounts of ultraviolet light radiation. He then measured the protein that would consequently be produced. By the summer of 1966, the new approach also failed.

Meanwhile, Walter Gilbert and Benno Müller-Hill, who worked in the same building at Harvard, had succeeded in isolating the lac operon’s famous repressor! Their lac repressor isolation work was published in December of 1966. James Watson reluctantly communicated the manuscript to the PNAS, knowing full well that Ptashne was so close to succeeding in purifying the λ repressor. It was Müller-Hill and Gilbert, however, who would be forever celebrated as having accomplished one of the most meaningful advances in the history of gene expression regulation.

Ptashne would be consoled from these previous failures, however, with the arrival at Harvard of a new laboratory technician, Nancy Haven Doe (later Hopkins) from Yale. At Yale, Hopkins had been an unhappy graduate student. Hopkins, who had been a Harvard-Radcliffe undergraduate student, now sought Watson’s help in coming to Harvard to work on the λ repressor. Hopkins felt she would be happier as a Harvard technician purifying the λ repressor than a Yale graduate student not studying the repressor.

Ptashne and Hopkins conducted the following, now classic, experiments to discover the bacteriophage λ repressor. They cultured the host Escherichia coli bacteria and irradiated them with massive doses of UV radiation. The radiation treatment destroyed DNA and minimized confounding extraneous protein production. Then, to maximize λ repressor production, Ptashne and Hopkins added bacteriophage λ virus to the Escherichia coli. They used a series of bacteriophage viral strains during infection to help suppress the production of unwanted non-λ repressor viral proteins.

They divided the phage-infected bacteria into two culture flasks. Each flask had a mixture of phage λ and bacteria. To one flask, they added the amino acid leucine that was labeled with a radioactive tritium tracer, leucine-[3H]. The other flask was provided with leucine tagged with radioactive carbon-14, leucine-[14C]. The bacteria-phages contents of both flasks were centrifuged, washed with buffer, and mixed. Thus, the two differentially labeled phage-bacteria sets, each with leucine-[3H] or leucine-[14C], were now combined. The two radioactive phage-bacteria mixtures were treated by sonication to destroy the host bacteria. The radiation numbers present in bacterial cellular contents were then measured.

In cells with wild-type λ phage, cells with leucine-[3H] or leucine-[14C] showed spikes in radioactivity compared to cells with mutant phage λ viruses, such as a mutant called CIsus34. In another experiment, Ptashne and Hopkins added a minimal amount of magnesium, which prevented radioactive leucine from being incorporated into a new protein. With high amounts of magnesium, the newly made proteins would contain radioactive leucine. The results showed that with high magnesium concentration, the repressor’s production could be turned on. This increase in detectible radioactive protein helped Ptashne and Hopkins on the next steps toward λ repressor purification.

Ptashne and Hopkins acquired batches of cellular extracts with labeled putative λ repressor. Then, they subjected the mixture to a diethyl-aminoethyl (DEAE) chromatography column, which bound the newly made viral λ repressor protein. The desired protein stayed attached inside the DEAE column. Next, they washed the DEAE column to eliminate any unwanted or contaminating proteins. Ptashne and Hopkins then eluted the bound λ repressor with a high salt solution to knock the protein off its place in the DEAE column. Next, Ptashne and Hopkins ran the eluted λ repressor protein on an electrophoresis apparatus. Then they counted the radioactivity.

They observed nice single peaks of leucine-[3H] or leucine-[14C] from bacteria that were infected with wild-type phage λ, but not in cells with mutant or inhibited phages. He called the pure protein “CI,” in keeping with the terminology of the great Jacques Monod. The single peaks of radioactively labeled protein strongly indicated that Ptashne and Hopkins had finally succeeded in purifying the soon-to-be-famous λ repressor!

Ptashne quickly wrote up the manuscript and arranged for submission to the PNAS on December 27 of 1966 to announce CI’s discovery, the λ repressor. The paper was accepted readily and published in February of 1967. There was an intense rivalry between the Gilbert lab and the Watson lab to discover a repressor protein. Ptashne would be close but would lose in being the first at Harvard to purify a repressor protein. Gilbert and Müller-Hill had beat Ptashne by two months! Interestingly, at the time, it was not customary for laboratory technicians to be considered for co-authorship in scientific publication, and Ptashne was, thus, the sole author. Hopkins was mentioned in the acknowledgments section for her excellent assistance.

3) The focus of most of Ptashne’s scientific career has been gene regulation. Why is this significant in the big scheme of things?

Before Ptashne had ever purified and identified the CI λ repressor protein, its presence had already been postulated. Jacques Monod had called the gene encoding the repressor “cI.” The cI repressor gene was thought to play a role in controlling “immunity” to phage infection, a lysogeny process. On the one hand, the lytic phase of the λ phage would kill the bacterium by lysing it. On the other hand, the lysogenic stage of phage λ kept the bacterium alive. Thus, in a sense, the bacterium was “immune” to phage infection. In today’s parlance, molecular biologists like to avoid the term “immunity” to denote the lysogenic stage. The immune system is distinct from the bacteriophage infection of bacteria.

After the significant discovery of the CI λ repressor protein by Ptashne and Hopkins, in 1967, he and his laboratory would spend decades figuring out how the repressor functioned. It was a massive undertaking involving unprecedented investigative effort and dozens of graduate students and postdoctoral fellows.

File:Phage Lambda SwitchStates.jpg

https://commons.wikimedia.org/wiki/File:Phage_Lambda_SwitchStates.jpg

Figure 74. Diagram of the phage lambda (λ) promoters RM and R and operator regions that affect the lysogenic or lytic states.

The λ repressor, called the CI protein or the “immunity substance,” controls the lytic process by repressing phage λ virus production. See Figure 74. The CI λ repressor protein binds to operator sites, called OL and OR1, OR2, and OR3. It was later found that these operator sites functioned to enhance the cooperativity of the repressor binding to DNA. The λ repressor blocks the operator sites on DNA. RNA polymerase binds the DNA but cannot move past the R promoter. Thus, the lytic genes are turned off. However, the RNA polymerase can work with the RM promoter, though the outcome is merely more λ repressor production. In this state, the λ repressor is functional. The lytic cycle is thus turned off, and lysogeny predominates. Therefore, the bacterium lives and is called a lysogen.

Sometimes, the lysogen is induced, where the phage goes lytic, and the bacterium dies. This phage induction is one aspect of the genetic switch—converting from lysogeny to the lytic state. The provocation involves the destruction of the λ repressor. The destruction process starts with UV light, which activates a protein called RecA. The RecA protein cleaves the CI λ repressor into pieces, inactivating it. The loss of the λ repressor permits the binding of Cro. This protein inhibits the CI λ repressor from binding to the operator and allowing RNA polymerase to turn on transcription of λ phage genes to make RNA and proteins. Production of λ phages then results in the lytic process.

In later years, the structure of Patashne’s CI λ repressor protein was deduced. It was shown to bind to the operator site on DNA. See Figure 75. In the diagram, the ribbons, shown on top, represent the protein’s helix-turn-helix domain of the CI λ repressor protein. In contrast, the DNA is shown at the bottom.

File:Lambda repressor binding DNA.png

https://en.wikipedia.org/wiki/File:Lambda_repressor_binding_DNA.png

Figure 75. Lambda CI repressor bound to DNA.

Another fascinating feature of gene regulation was discovered, and it involved the looping of DNA! See Figure 76. In this scenario, Ptashne and colleagues found that the λ repressor sites were numerous and distantly located elsewhere on the DNA. There were two sets of operators, on the left and the right of the cI gene. The question arose as to how the λ repressor could bind such distantly located regions on DNA to mediate their gene regulation behavior.

The answer came to Ptashne in the form of “DNA looping.” The DNA with their bound λ repressor proteins turned around, forming a loop and interacted! These types of DNA-protein interactions permitted a sort of cooperation between them to enhance gene regulation activity. The phenomenon was called cooperativity. It was a fantastic discovery that is still treated in molecular biology textbooks.

File:LambdaPhage Repressor Cooperativity.jpg

https://commons.wikimedia.org/wiki/File:LambdaPhage_Repressor_Cooperativity.jpg

Figure 76. Visual representation of DNA looping with repressor tetramer/octamer binding to phage lambda L and R operator sites (stable lysogenic state).

The work of Ptashne led to the discovery of the famous λ repressor and elucidation of its genetic switching mechanisms. Ptashne and collaborators elucidated how phages and bacteria permitted the switching between gene expressions. The genetic switch studies would be hailed as part of a significant scientific breakthrough in gene expression regulation. The genetic switching enabled the conversion from lysogeny and the lytic process. Such mechanistic knowledge of gene expression switching opened the floodgates of molecular biology. New scientific advances were now possible. The development of new chemotherapies might be possible. Gene cloning technology was in its infancy. The gene expression regulation systems allowed protein production to be turned on or off when necessary. This early work would also lead to the analysis of mutants, sequencing technologies, gene therapy, and genomic editing inventions.

4) Apparently, Ptashne did something quite unorthodox at the time—he founded in 1980 with a colleague Thomas Maniatis something called the Genetics Institute. Now, what was he trying to accomplish, and why did this “ruffle some feathers” at Harvard?

Later, in 1980, Ptashne chaired the Biochemistry and the Molecular Biology department. Tom Maniatis arrived at Harvard and worked under Ptashne as a postdoctoral fellow. In Ptashne’s lab, Maniatis would acquire gene-cloning expertise. Maniatis collaborated with Argiris Efstratiadis, who was a postdoctoral fellow from the lab of Fotis Kafatos. Together, they focused on the gene for the β-globin protein called factor VIII, which worked in erythrocytes of red blood cells. Soon, Maniatis became a faculty member on the tenure track at Harvard. At some point, protests against recombinant DNA technology were transpiring and becoming more vocal in their disapproval. The “feather-ruffling” had occurred both on the campus of Harvard as well as in the township of Cambridge, MA. Maniatis thus moved to Cold Spring Harbor with James Watson, who was its new leader.

In 1980, Ptashne and Tom Maniatis co-founded a biotechnology company called Genetics Institute, colloquially called “GI,” and is now a subsidiary of Wyeth Pharmaceuticals. At the GI, Ptashne and Maniatis got on with cloning the gene for factor VIII and getting cell systems to produce the protein in large quantities. It was a substantial undertaking because the factor VIII protein contained 2,350 amino acids. By molecular standards, the protein was colossal.

To further complicate matters, the factor VIII gene had dispersed within its specific introns. The introns were intervening sequences that had to be removed first before the mRNA could be translated into factor VIII protein. To get around this problem, they tried an innovative approach. First, they prepared mRNA from animal cells. Next, they used the enzyme called reverse transcriptase to produce double-stranded DNA. Fortunately, the mRNA and its reverse-transcribed DNA lacked the confounding introns! The newly made DNA encoding the factor VIII protein contained only the exons, i.e., the gene’s expressed segments remained intact. Now the copied DNA could be cloned into a plasmid DNA vector and expressed in hamster cells. It permitted the production of factor VIII in host cells, from which the protein could be extracted. It was a successful advance in molecular biology and recombinant DNA technology. The protein could be used clinically as a blood clotting factor in patients who needed it. Fundamental was the fact that the clotting factor VIII could readily be made without confounding human blood contamination.

It was getting his GI company established that had brought Ptashne considerable gratification. He made several million dollars; though, he is not presently a member of this company. Ptashne also co-founded a new biotechnology company called Acceleron Pharma. Both companies are located in Cambridge, Massachusetts. In 1993, Ptashne was appointed as the endowed Herchel Smith Professor of Molecular Biology.

5) Currently, Ptashne is the Ludwig Chair of Molecular Biology at one of the world’s most famous cancer centers—Memorial Sloan-Kettering in New York. Do you know his charge there or what he was trying to investigate?

Ptashne’s new challenge arrived in 1997 when he accepted the Ludwig Chair of Molecular Biology at Memorial Sloan-Kettering Cancer Center in New York. He is primarily focused on studies of the gene regulation mechanisms in yeast microbes.

He studied gene regulation for galactose metabolism (a sugar) in yeast, called Saccharomyces cerevisiae.Before the microbes are fed galactose, nucleosomes are stuck on the DNA elements called promoters that control the galactose metabolism genes GAL1 and GAL10. After the yeast cells are provided the sugar, a DNA-binding activator protein called Gal4 is bound to a site called UASg, meaning an upstream activating sequence for galactose. Gal4 then recruits the enzyme called SWI/SNF, which stands for SWItch/Sucrose Non-Fermentable protein. The SWI/SNF enzyme removes the nucleosomes from the promoters. The nucleosome removal permits the expression of the GAL1 and GAL10 genes so that galactose can be metabolized.

Another gene regulation system that was reminiscent of the DNA looping mechanism of λ repressor was the so-called kit locus on murine DNA. This time, however, the DNA sequences that came together by a DNA looping mechanism involve the promoter for the kit gene and an enhancer element. The looping of the DNA segments permits the removal of bound nucleosomes so that so-called activator proteins can now bind. The enabled RNA polymerase initiates transcription of the kit gene, effectively turning it on.

Some of Ptashne’s most recent work involves converting adult human and mouse cells into induced pluripotent stem cells. The new work uses a variety of transcription factor proteins to mediate this new cellular reprogramming. The research is significant because the cellular reprogramming system involves using enhancer elements and transcriptional activators working in conjunction with each other. The system can circumvent embryonic stem cells, a controversial practice, by exploiting already differentiated adult cells to induce the cell reprogramming method.

6) Like many other scholars—Ptashne was awarded several prizes and wrote several books—his most important in your mind?

Ptashne was bestowed the N.A.S. Award in Molecular Biology (1979), the Louisa Gross Horwitz Prize (1985), the Lasker Award (1997), and the Massry Prize (1998). Frequently, historians of science like to argue that the Lasker Award is the precursor to the Nobel. While it may infrequently be the case that the Lasker Award precedes the Nobel, such was not in the offing for Ptashne. The lack of a Nobel for Ptashne may reflect that his isolation and mechanistic studies of the λ repressor were not the first to be discovered or elucidated. That distinction can go to Jacques Monod, André Lwoff, and François Jacob for their ingenious discovery of the lac repressor and its accompanying lactose-metabolizing genes lining the lac operon.

One of Ptashne’s writings is considered a classic, if not a delightful little book. The book is titled “A Genetic Switch: Gene Control and Phage λ.” One of us (M.F.V.) recalls that in the late 1980s, Ptashne’s book was required reading in a molecular biology course in graduate school. We all thoroughly enjoyed the classic work. I recall that it was the first time that any of us in the class had heard of Mark Ptashne. As we discussed the book, we also speculated on how to pronounce “Ptashne.” About half of us left out the “P” sound.

7) What have I neglected to ask about this molecular biologist?

Ptashne is a skillful musician and has studied the violin privately with expert music instructors. He participated in recording a musical CD, an excerpt of which is included at the end of this chapter.

Each Christmas, at Harvard, the faculty are customarily roasted. While most professors justifiably dreaded mockery, Ptashne’s main worry was that he might be passed over. One skit that had been shared involved him, a photocopier, and a departmental secretary. He seemed disappointed and complained that the story was from the past and already familiar to those in attendance. There was, he professed, “a lot of newer and better material they could have used.”

Click on the following links to acquire supplementary information on this remarkable pioneering molecular biologist.

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