Stanley Norman Cohen and His Contributions to Molecular Biology An Interview with Manuel F. Varela and Ann F. Varela

Oct 24, 2020 by

Stanley Norman Cohen

Michael F. Shaughnessy

1) Born in New Jersey and on to greatness—When was Stanley Cohen born, and where did he go to school?

Stanley Norman Cohen is best known for his involvement in the inventions of DNA transfer, bacterial transformation, gene cloning, and the resulting emergence of biotechnology. His birth year was 1935. Stanley N. Cohen was born into the home of Bernard and Ida Cohen on June 30 in Perth Amboy, New Jersey. Cohen’s grandparents were Jewish immigrants. Cohen’s father, Bernard, was employed as an electrician while his mother, Ida, made a living with her secretarial and bookkeeping skills.

Ten years after Stanley’s birth, a baby sister, Wilma, joined the family. In his youth, Cohen was most interested in how things worked, especially in electronics and science. Cohen often assisted his father with supplemental endeavors, which were meant to increase the family income. One such activity involved the manufacture and peddling of fluorescent light fixtures, transformers, and electric fans.

Cohen attended Rutgers University to pursue a career in medicine. He earned his B.S degree in 1956 in the area of Biological Sciences. In 1956, Cohen began his medical school studies at the University of Pennsylvania, School of Medicine. While there, he partook in a laboratory venture studying immunological rejection of foreign skin grafts.

Charles Breedis, in the Department of Pathology, was his supervisor. This project led to a collaboration with Rupert Billingham at the Wistar Institute. In 1959, Cohen became a student in the laboratory of Peter Medawar in London. In 1960, Cohen completed all requirements for his medical degree. For two years, 1960-1961, Cohen was an intern at Mount Sinai Hospital. After that, he engaged in two years of residency in internal medicine at the University of Michigan.

2) The institutions where he did research—what do we know about Cohen’s career?

Cohen worked as a Public Health Service officer at the National Institutes of Health (NIH), where he combined basic research with clinical medicine. His field of study is similar to an M.D./Ph.D. program today. It was during this time at NIH that Cohen was one of many physicians drafted to satisfy military duties associated with the Berlin Wall crisis in 1961. Cohen worked in K. Leome Yielding’s laboratory investigating the interaction between the anti-material drug chloroquine and DNA.

Cohen worked on his clinical training as a senior resident from 1964-1965 at Duke University Hospital in Durham, North Carolina, and then spent two years at the American Cancer Society as a post-doctoral fellow until 1967 at Albert Einstein College of Medicine in Bronx, New York. His experiments dealt with lambda bacteriophage.

Cohen joined the faculty of Stanford University in 1968 as Kwoh-Ting Li Professor of Genetics and Professor of Medicine in order to continue his study of plasmids. There he persevered to develop his interest in the resistance of bacteria to antibiotics. Cohen did this research in conjunction with helping to set up the Division of Clinical Pharmacology. He directed this division until he moved to the Department of Genetics in 1978.

Cohen met Herbert Boyer in 1972. The two talented and innovative researchers invented DNA cloning methods.

3) Plasmid DNA—seems to be a key element of his work at Stanford. What exactly is this, and why is it important?

Plasmids are tiny circular pieces of DNA carried by microorganisms and replicate independently of the host genomes. Plasmids also can carry various genetic determinants that confer new properties which permit specific advantageous characteristics to hosts harboring them. These plasmid molecules can confer, in some cases, fermentation of a nutrient, or become resistant to an antimicrobial agent, or even transfer of DNA to another host.

Plasmids can be naturally occurring in the environment, protected by larger hosts, such as bacteria, or fungi, for instance. Many plasmids are able to carry out DNA replication using molecular mechanisms that function separately from the DNA synthetic machinery of the host cells that carry the plasmids. Thus, an investigator can add an inhibitory substance that prevents genomic DNA replication but nevertheless permits plasmid DNA to replicate unhindered.

Cohen and his collaborators exploited plasmid molecules to exchange genetic elements and form recombined versions of different plasmids to create novel ones, which in turn could be used to clone genes from all forms of life. Thus, because of Cohen’s expertise with plasmid biology, one could clone new genes, and these efforts permitted molecular biologists to examine genes more closely at the molecular level and to study gene expression and regulation. Furthermore, gene cloning allowed investigators to create new variants of genes in order to study their structure-function relationships in proteins encoded by the parental genes. The plasmids could be linearized with restriction endonucleases. See Figure 22. Alternatively, the plasmids could remain in a so-called close configuration, but nicked such that the circular plasmid opened up, or kept in an intact circle, making it coil upon itself in a so-called supercoiled form. See Figure 22.

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Figure 22. Plasmids in different shapes under an electron microscope.

In more recent times, the knowledge gained by gene cloning has facilitated the progress in the fields of genomics and biotechnology. Gene cloning permitted investigators to compare gene and genome sequences, with the advent of DNA sequencing technologies, starting in the late 1970s. Then, with new advances in the generation of mutations and isolation of new mutant organisms, one could explore the relationship between protein structure and their biochemical functions.

Later, in the early 21st century, the new genome editing technologies, such as those developed by recent 2020 chemistry Nobel Laureates, Drs. Emmanuelle Charpentier and Jennifer A. Doudna, has made it possible to alter the genes of living organisms.

Each of these advances in molecular biology was made possible by the pioneering work of Stanley N. Cohen and his knowledge of the biology of plasmid DNA.

4) Transferring DNA from organism to another—what was Cohen’s involvement, and what was he researching?

In the latter part of 1973, Stanley Cohen worked with Annie Chang at Stanford and with Herbert Boyer and Robert Helling at the University of California at San Francisco. Together, the two teams of investigators participated in the first successful transfer of DNA from one form of life to another completely different life form. The collaborative work would eventually lead to the development of molecular gene cloning and, thus, of the invention of recombinant DNA technology. See Figure 23 for an overview of the molecular cloning process.

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Figure 23. Molecular Cloning.

This early groundbreaking DNA transfer work involved connecting a region of DNA from a plasmid of one bacterium to the section of plasmid DNA from another bacterium, creating a hybrid DNA variant with altered antibiotic resistance profiles. Such a genetic combination in the laboratory setting had never been accomplished before, and it was Cohen’s knowledge of plasmid DNA biology, which would facilitate the progress. In short, the Stanford and UCSF teams would, for the first time in history, artificially create a new plasmid DNA molecule, and it would be one of the world’s first recombinant DNA molecules ever made by human beings.

Next, Cohen and his co-workers transformed host Escherichia coli cells with their newly formed recombined DNA and let the bacteria grow new colonies on culture media. The mode of transformation was experimental. Prior to this, the transformation phenomenon was known to occur naturally. Cohen and lab colleagues used a chemical called calcium chloride (CaCl2) to make the Escherichia coli artificially “competent” to acquire foreign DNA molecules. Once the host bacteria fully took up the recombined DNA. The resulting transformants now had resistance to multiple antibiotics, such as tetracycline, the gene of which had come from pSC101, and resistances to kanamycin and chloramphenicol, the determinants of which had arisen from plasmid R6-5.

The artificial transfer of newly recombinant DNA molecules from the laboratory bench (see Figure 24) to a host organism, like Escherichia coli, would facilitate the elucidation of new methods. Chemical transformation permitted new advancements in gene cloning. Other advances in molecular biology became possible, such as site-directed mutagenesis, sequencing, regulation of gene expression, and genome editing.

5) He did some research on “recombinant DNA” and Escherichia coli. What exactly did he do, and what did he find?

Cohen’s work led to the discovery of the world’s first recombinant DNA molecules with bacterial resistance genes. His work involved the creation of new combined DNA segments, each harboring different resistance genes from different bacterial isolates. The work would lead to a revolution in molecular biology, biotechnology, recombinant DNA.

While the technical details are described in the previous chapter on Boyer, the research collaboration used DNA from two distinctive naturally occurring plasmid DNA molecules that had originated from different strains of Escherichia coli. One of these plasmids, called R6-5, had been discovered by Tsutomu Watanabe, Chizuko Ogata, and Sachiko Sato, in their laboratory at Keio University, Tokyo, Japan. The R6-5 plasmid had genes for resistance to kanamycin and chloramphenicol encoded within its DNA. The other plasmid, pSC101, harbored a tetracycline resistance determinant and was isolated by Cohen and Chang from another Escherichia coli strain. Cohen and colleagues cut the two plasmids using newly discovered and purified restriction enzymes, mixed various DNA fragments from R-65 to restricted pSC101, and ligated the various DNA pieces with ligase enzyme. Cohen and his group published the pioneering work in November of 1973.

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Figure 24. Stanley Norman Cohen’s Genetic Engineering Laboratory, 1973 – National American History Museum’s Science in American Life exhibit.

Another study was conducted shortly after their historical work was published. Cohen and Annie C. Chang next recombined DNA molecules with plasmids from entirely different species of bacteria. This time, they considered a plasmid that was isolated from Staphylococcus aureus, a Gram-positive bacterium, whose variants were capable of serious infectious disease. The other DNA molecule that they considered was the pSC101 plasmid that had been successfully used to clone antimicrobial resistance genes in their previous study.

Using their gene cloning tools now available to them at their disposal, Chang and Cohen cloned resistance genes from the pI258 plasmid DNA of Staphylococcus aureus into pSC101, now referred to as a cloning vector. The resulting recombinant DNA molecules had its genetic origins from two different species of bacteria, Staphylococcus aureus and Escherichia coli. Chang and Cohen had cloned staphylococcal genes from the pI258 encoding resistance to the antibiotics like penicillin and erythromycin, and resistance to heavy metals, such as mercury and cadmium. The newly cloned genes, generated artificially by recombinant DNA techniques, were used to transform freshly cultured Escherichia coli host cells to closer study the new resistance behavior on Petri dishes. The new findings were published in April of 1973.

The new recombinant DNA tools could be invoked for generating DNA hybrids with other bacterial species, such as between Escherichia coli and Salmonella enterica, serovar Panama, which at the time, was initially called Salmonella panama.

In this study, Cohen and Chang cloned a tetracycline resistance gene from the Salmonella enterica and expressed the new resistance properties to recipient Escherichia coli host cells after chemical transformation. The new work was published in May 1973.

Elements of Cohen’s genetically engineered DNA molecules would lead to widespread gene cloning, especially with the creation of the popular cloning vector, called pBR322. See Figure 25.

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Figure 25. pBR322.

The pBR322 cloning vector, with its array of unique restriction sites, could be used to insert new DNA restriction segments, ligated, and transformed into host bacteria for propagation. The reporter genes, such as those for resistance to tetracycline or penicillin (or ampicillin), could readily indicate whether gene cloning attempts were successful. It became a plasmid of choice for molecular cloning throughout the 1970s and 1980s.

As discussed in the previous chapter, Cohen, Boyer, and colleagues would move to the recombination of different DNA segments from prokaryotes and the higher organisms, such as fungi. This type of recombinant DNA technology was a new advance. Yet, the combining of DNA from species of bacteria had become alarming.

6) Cohen’s work with Herbert Boyer ended with a call for a moratorium on genetic engineering—What happened?

At the time, in the early 1970s, that Cohen and collaborators had figured out how to successfully recombine DNA from different species of living organisms, concerns began to be raised about the ramifications. The tensions had been raised when the news spread about genetic engineering tools now seemingly freely available in the laboratory.

Such concerns were evident after the publication of Michael Crichton’s fiction novel “The Andromeda Strain,” and the film of the same name that had been inspired, in 1968. Crichton had speculated about contamination of a space satellite returning to Earth with a newly mutated microbe that caused a severe pandemic. In Crichton’s book, radiation exposure in outer space of the microbe rendered it highly virulent. Upon its return to Earth, the fictitious Andromeda strain wreaked havoc on individuals, causing painful and immediate death in those who were exposed to it. While the book and film were purely speculative, it nevertheless raised the eyebrows of people who took such cases as potentially possible, especially now that alteration of genetic material became possible in the laboratory.

The published works on the molecular biology by Stanley N. Cohen, Herbert Boyer, Paul Berg, and others led to raising serious new concerns regarding similar types of infectious hazards that might consequently be possible with the prospects now available with genetic engineering. Critics wondered whether recombining DNA molecules from different species of organisms might result in unanticipated consequences. Could genetically engineering new DNA hybrids lead to the creation of potentially lethal, if not seriously virulent, and perhaps easily transmissible, contagions being inadvertently let loose upon the world outside the laboratory?

Investigators from various scientific disciplines were similarly concerned with the rapidly advancing genetic engineering field. The new biotechnological questions were also relevant to scientists in virology, genetics, biochemistry, microbiology, molecular biology, and medicine. Experts in each of these scientific fields held a meeting at the Asilomar Conference Center, near the Monterey coast in California, in the U.S. The first meeting, called “Asilomar I,” in January of 1973, was startling. The list of potential dangers to humankind was terrifying, especially in the light of what was not known yet about the particulars of the new genetic engineering field. In the early 1970s, it was an essentially brand-new field of study, and much about it was not yet fully understood.

With such frightening possibilities seemingly at reach within the confines of a simple laboratory bench, the Asilomar I conference attendees had no choice but to declare a moratorium on new experiments with genetic engineering until more could be learned about the details of cloning, DNA splicing, transformation, genetic manipulation, and the like.

In 1975, another conference was held at the same Asilomar Conference Center. At the new meeting, which has been referred to as “Asilomar II,” the attendees (including Cohen and others from all over the world) were supposed to hammer out a series of guidelines to implement. But the conference soon became chaotic. Instead of deliberating the next steps for policy, the attendees questioned whether a moratorium was necessary. Some attendees felt that scientific endeavors should not be so restrictive so as to hinder critical new advances in biomedicine and medicine, or even of basic scientific research.

At Asilomar II, tempers flared, and voices were raised. The meeting now consisted of name-calling, threats of litigation, and dramatic walkouts. One source reported that some of the attendees left the meeting in disgust, went to the beach, and smoked marijuana. Especially sobering were the legal ramifications inherent in the accidental release of a lethal genetic recombinant, as outlined by the lawyers attending the conference. They spoke about the potential for permanent closures of laboratories and entire institutions. Scientific research itself was at stake.

On the last night of the Asilomar II conference, the attendees realized that if they didn’t agree to the implementation of new laboratory policies, the politicians and lawyers who knew next to nothing about genetic engineering’s intricacies would step in and perhaps establish prohibitively draconian policies. Thus, the investigators took it upon themselves to formulate a few formal but simple policies for the prevention of the release of dangerous genetically engineered microbes.

Containment was imperative. Special biological hoods, with high capacity filters and outfitted with special ultra-violet lamps for sterilization of benches, were recommended. The molecular biologists also agreed to work with microbes that could not grow outside the laboratory without special supplementation in their culture media. Any escaped microbe would soon die if out of the lab. Lastly, it was agreed that prior to performing any new genetic engineering experiments, new proposals would be first reviewed and approved by panels of experts in the fields of interest.

With hindsight, it is readily clear that the laboratory measures taken have been successful. No genetically engineered mutants have ever wreaked havoc on society, despite the dubious claims otherwise, even with SARS-CoV-2 and the COVID-19 pandemic. Current evidence does not support the outlandish claim by conspiracy theorists that SARS-CoV-2 was an artificial mutant that escaped the laboratory. No such credible evidence supports that false claim. On the contrary, mounting evidence supports the conclusion that tSARS-CoV-2 is not an escaped genetically engineered mutant.

7) What have I neglected to ask about this scientist from New York?

As of this writing, Cohen is alive. He enjoys skiing, hiking, playing the five-string banjo, and sailing on Genesis, his boat. Cohen is also a pop music composer, writing under the pen name of Norman Stanton. He wrote a song called “Only You” in 1953.

Cohen acquired various awards for his involvement with the advancement of genetic engineering. Some of his awards included the Albert Lasker Award for Basic Medical Research (1980), the National Medal of Science (1988), the National Medal of Technology (1989), the Albany Medical Center Prize (1994) shared with Boyer, the Shaw Prize in Life Science and Medicine (2004), and the Double Helix Medal (2009).

Stanley Norman Cohen (b. 1935) should not be confused with other investigators of the same last name.

For example, Seymore Stanley Cohen, born in 1917, was noted for his work on phage biology. Seymore S. Cohen was born in New York, went to the City College and Columbia, and took his Ph.D. in 1941. Dr. S.S. Cohen was a faculty at the University of Denver, Colorado, in the U.S. Seymore S. Cohen made groundbreaking studies of the so-called burst size of T2 phages. S.S. Cohen also traced radioactive phosphorous through nucleic acids of the phages during infection of bacteria. S.S. Cohen’s work would later be famously linked to the historical experiments of Alfred Hershey and Martha Chase.

Another namesake was Stanley Cohen, born in New York in 1922. He would earn the Nobel Prize in physiology or medicine along with Dr. Rita Levi-Montalcini, for their discovery of nerve growth factors and their receptors.

For more information about Dr. Stanley Normal Cohen, visit the Link:

Discography: https://www.discogs.com/artist/3715023-Norman-Stanton

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