In the early 1970s, a momentous series of events in the history of science unfolded at points around the San Francisco Bay. Lines of inquiry pursued at the Stanford University School of Medicine and the University of California, San Francisco converged on a set of discoveries that vastly expanded the productive capabilities of molecular genetics, disrupted the customary rhythms and routines of the scientific community, sparked bitter disputes about risks and responsibilities in scientific experimentation, and generated a tsunami of technological change that spread rapidly across multiple domains of productive activity and all around the globe.
The first recombinant molecule containing DNA from different organisms was assembled late in 1971, in Paul Berg’s laboratory at Stanford. Berg hoped to transduce bacterial and mammalian cells with a recombinant virus in order to study gene expression systems, but subsequently chose not to carry out the planned experiments. He was persuaded by scientific colleagues to consider potential biohazard risks before moving ahead.
The technology for propagating and expressing recombinant genes was invented by Stanley Cohen and Herbert Boyer in 1973. It enabled the transformation of bacterial cells into living factories for the directed manufacture of select proteins. The technology was immediately recognized as a tool without parallel in genetics research, and was soon applied to practical ends in a wide variety of fields including medicine, pharmaceuticals, agriculture, chemicals, and energy. It has since transformed the world in which we live.
The history is complicated. The episode unfolded in an environment characterized by enabling collaboration but also intense competition. The principal coin of the scientific realm is recognition — credit for contributions and priority in discovery. When stakes are high, allocations are often controversial. In the case of recombinant DNA technology, disagreements persist about who made the crucial contributions, and who was or was not properly recognized. For formal scientific and legal purposes, the record is well-defined, but the participants are not in full accord about the timing of certain events and the authenticity of certain accounts. The currently available body of evidence doesn’t answer all outstanding questions. More historical research is needed.
Below, we trace what is known about the key technical developments through biographical accounts of the lead actors. In the early 1970s, Berg was already established as a scientific star, the heir apparent to the academic fiefdom of his mentor, Nobel Prize-winning biochemist Arthur Kornberg. Boyer and Cohen labored in relative obscurity in distant provinces of microbial genetics. The three men had not a lot in common, and they came into their historical roles on disparate paths, but as the story of the invention reveals, each was uniquely prepared and positioned to shape the course of events.
PAUL BERG
Paul Berg was born in Brooklyn in 1926, the son of immigrants, Russian Jews from a small village near Minsk. His parents had no formal schooling, but they placed a high value on education. As a youth, Berg was encouraged to achieve academically, and after reading Arrowsmith by Sinclair Lewis and The Microbe Hunters by Paul de Kruif, he developed an abiding interest in biomedical research.
His high school grades qualified him for a full scholarship to the City College of New York. He majored in biology, but quickly became bored by the classical curriculum. He decided to leave, and ended up at Penn State after a friend from Brooklyn sent a brochure on the school’s biochemistry department. “It wasn’t chemistry and it wasn’t biology,” he says, “but it looked like what I was interested in — the processes that go on in living organisms.”
Like many of his contemporaries, Berg’s college career was interrupted by a three-year tour of military duty at the end of World War II. He trained as an ensign in the US Navy, and served on subchasers that escorted shipping convoys in the Atlantic Ocean and the Caribbean Sea. Following the surrender of Japan, he was reassigned to the Pacific. For a year, he worked on crews that returned naval ships to the United States from far-flung outposts in Asia. Afterwards, he went back to Penn State to complete his undergraduate studies.
When summer vacations approached, Berg wrote dozens of letters soliciting employment from companies in the New York metropolitan area. He took temporary jobs at General Foods and the Lipton Tea Company. His experiences in industrial chemistry made an impression: “The people with bachelor’s degrees were being told what to do. The people telling them what to do had PhDs. That’s when I decided that I wanted a PhD.”
Berg did his graduate work at Western Reserve University in Cleveland under the direction of famed biochemist Harlan Wood. The department specialized in research conducted with radioisotopes. Berg made an early name for himself by using radiolabels to track amino acids and vitamins through the biochemical reactions that constitute intermediary metabolism. He showed that methionine and choline are not essential dietary requirements, as experts insisted, but are manufactured by the body given adequate supplies of folic acid and B-12.
When Berg finished his PhD, Wood wanted to place him in a post-doc at Washington University in St. Louis with revered biochemists and Nobel Prize winners, Carl and Gerti Cori. Berg preferred Copenhagen, however, and went to work for a year in Herman Kalckar’s laboratory at the Institute of Cytophysiology. There, in collaboration with an Australian named Bill Joklik, he discovered an enzyme called NDPK (nucleoside diphosphokinase) that is involved in fundamental processes of nucleic acid biosynthesis and metabolism.
For a second post-doctoral year, Berg moved to Washington University, but not to the Cori lab. He joined the team surrounding an up-and-coming star, enzymologist Arthur Kornberg, and again performed a deft bit of biochemistry. Against conventional wisdom, he proved the existence of another key component of basic cellular metabolism, a molecule called acetyl-AMP (adenosine monophosphate). The elegant work earned him the admiration of top researchers in the field.
Berg stayed on after his post-doc, at Kornberg’s request, in a temporary appointment, a bridge to a full-time faculty position. Kornberg discovered DNA polymerase in 1956, and his laboratory focused increasingly on the biochemistry of DNA replication. In 1959, Kornberg received a Nobel Prize for DNA polymerase, and moved his entire group, including Berg, from St. Louis to Stanford.
At the same time, bacteriologist Joshua Lederberg relocated to Stanford from the University of Wisconsin, to establish a department of genetics. Lederberg also owned a Nobel Prize. With Edward Tatum, he discovered in 1946 that bacteria acquire genetic information through conjugation, the exchange of plasmids (circular loops of functional DNA) between bacterial cells that come into physical contact. In 1951, he and Norton Zinder documented the phenomenon of transduction, the insertion of foreign DNA into bacterial cells by viruses. Following the arrival of Kornberg and Lederberg, bits and pieces of recombinant DNA technology began to collect at the Stanford University School of Medicine.
In 1967, Berg stepped away from pure enzymology toward a program with broader implications for molecular genetics. He became interested in gene expression and regulation in mammalian cells, and took a sabbatical to study tumor viruses with Renato Dulbecco at the Salk Institute for Biological Studies in La Jolla, California. Kornberg was infuriated. He believed that progress in biochemistry and genetics would be achieved most directly by working to understand E. coli, one of the life sciences’ key model organisms.
After decades of research on the bacterium, more was known about fundamental life processes in E. coli than in any other creature. In Kornberg’s opinion, research on simple (usually single-cell) prokaryotic systems would shine the brightest light on fundamental processes in eukaryotes — more complex multicellular organisms. Berg was not confident in the assumption and proposed to test it by experimenting with mammalian cells.
The senior scientist feared that his protégé would be waylaid by a confusing mélange of cellular artifacts. According to Berg, Kornberg told him, “You’re wasting your talent, destroying your career. You have a gift for doing enzyme research. The only true path to knowledge is E. coli.”
Berg began working with polyoma, a murine virus, and later with SV40 (simian virus 40), which infects primate cells. Modern molecular biology had been built on research with viruses. From the late 1930s through the 1960s, pioneers in the field formed a virtual network with central hubs at Caltech and Cold Spring Harbor Laboratory. Led by Max Delbrück, the ‘phage group’ as it was known, employed a virus called bacteriophage as a model organism in experiments that established the foundations of bacterial genetics. Much of what was known about E. coli had been derived from bacteriophage research. Berg saw SV40 as a similar instrument, a utensil for studying the regulation and expression of genes in mammalian cells.
SV40 is a delivery vehicle that introduces its own DNA into host cells. The viral genes then reproduce new virus particles or become incorporated into host chromosomes. Berg wanted to take advantage of this natural property to answer an important question: do the mechanisms that regulate gene expression in bacteria also function in eukaryotic cells? Few labs had taken up the question. To investigate, Berg wanted to turn SV40 into a transduction technology, a means of incorporating specific foreign genes into mammalian genomes. He wanted to make a customized particle of infectious DNA.
Back in Palo Alto, Berg had all of the right enzymatic tools for the job. He contends that the Stanford biochemistry department became the world leader in molecular genetics because it had access to Arthur Kornberg’s refrigerator. What Berg lacked was DNA. Fortunately, a neighboring lab run by Dale Kaiser possessed a good source, a plasmid called lambda dv gal that contained three E. coli genes in DNA from lambda phage, a bacterial virus.
Kornberg’s Refrigerator
As a force in the development of enzymology during the second half of the twentieth century, Arthur Kornberg (1918–2007) was peerless. He earned a share of the 1959 Nobel Prize in Physiology or Medicine with his former teacher, Severo Ochoa, for discovering fundamental mechanisms of RNA and DNA biosynthesis, and went on to amass a legendary wealth of scientific talent at Stanford in the Department of Biochemistry. Remarkably, the six original members of the department — Kornberg, Robert Baldwin, Paul Berg, David Hogness, Dale Kaiser, and Robert Lehman — stayed together for nearly fifty years.
By the end of his career, Kornberg had acquired sufficient authority to deliver “The Ten Commandments” (of enzymology) to the Journal of Bacteriology. Among the injunctions: “Trust in the universality of biochemistry,” “Thou shalt not waste clean thinking on dirty reagents,” and “Thou shalt not waste clean reagents on dirty substrates.”
Kornberg maintained that recombinant DNA first appeared in Palo Alto because of his department’s unparalleled knowledge and understanding of nucleic acid biochemistry. The refrigerator in his laboratory was stocked with the world’s premier collection of enzymes involved in processes of DNA transcription and translation. In a memoir on his life in science (For the Love of Enzymes: The Odyssey of a Biochemist), Kornberg wrote:
“Significant roots of genetic engineering grew at Stanford because we had discovered or were applying the reagents basic to the manipulation of DNA: polymerase to synthesize long chains of DNA and fill in gaps, ligase to join contiguous ends of chains, exonuclease III to remove obstructive phosphate groups at chain ends, phage λ exonuclease to chew back one end of a DNA chain, and terminal transferase, an enzyme (from the thymus gland) to add nucleotides, willy-nilly, to the other end of a DNA chain.”
David Jackson, the lead author on the original recombinant DNA paper, agrees with Kornberg’s historical assessment. Of Stanford in the early 1970s, he says:
“It was absolutely the right place at the right time, the only right place at that time. You could argue that Charlie Richardson’s laboratory at Harvard Medical School might have done it, but Charlie didn’t have access to all of the enzymes that were used to modify the substrate DNA, and he didn’t have access to all of the tremendous expertise that was present at Stanford. If you were at Stanford and wanted to talk to the world’s leading expert on E. coli ligase, there was Paul Modrich in Bob Lehman’s laboratory, who had purified the stuff and was the source of it. If you wanted to talk about the details of DNA polymerase, you could walk down the hall to see Doug Brutlag, the graduate student in Arthur’s laboratory who had purified large quantities of it. It was the same for λ exonuclease, the same for XO3 [exonuclease III]. It was just amazing. That concentration of knowledge and expertise could not be found anywhere else that I know of.”
Stanford’s centrality in subsequent recombinant DNA research resulted from synergies between the Department of Biochemistry’s expertise in enzymology and research programs in genetics underway — within the department and without — in the labs of Stanley Cohen, Ron Davis, David Hogness, Dale Kaiser, Joshua Lederberg, Charles Yanofsky, and others.
Berg wondered whether lambda dv gal could be integrated into rings of purified SV40 DNA. If so, the bacterial genes could then be inserted into mammalian cells for expression tests. David Jackson, a post-doc in Berg’s lab, began working to recombine lambda dv gal and SV40 DNA by attaching single-stranded chains of complementary nucleotides (the building blocks of DNA molecules) to the ends of each. He expected that the complementary single strands would combine naturally and form a bridge linking the two original molecules.
Jackson attributes the idea to Berg: “I think it was Paul who saw that you could add complementary tails to the ends of DNA molecules, and thereby have two structures with a built-in mechanism for linking.” Berg also credits Peter Lobban, a graduate student in the lab of Stanford biochemist Dale Kaiser, with conceptualizing the process. Lobban, he says, “independently conceived the idea of using a series of enzymes to covalently join DNAs together in vitro.”
Lobban outlined a method for recombining DNA in a 1969 proposal submitted as PhD qualifying exercise in advance of his thesis research on lambda phage. The critical ingredient in the method was an enzyme called terminal transferase which adds nucleotide bases to the ends of pieces of DNA.
It was Lobban’s idea to use terminal transferase to create a batch of bacteriophage P22 DNA molecules with single-stranded poly(A) tails attached to the ends, and another batch with poly(T) tails attached. Lobban hypothesized that when the two batches were mixed together, the complementary tails would hybridize. Finally, he proposed that DNA polymerases and ligases could be added to seal the joints.
Lobban adds to the genealogy of the approach: “I actually got the idea from a seminar given by Tom Broker, a fellow graduate student, who threw off a suggestive remark at the end of a journal club talk on terminal transferase. He mused that somebody might be able to use the enzyme to create cohesive ends. I remembered that.” At the time, Lobban was intrigued by the notion because he thought the action of terminal transferase might help to explain the genomic incorporation of viral DNA.
Poly(A) Poly(T) tailing
The poly(A) poly(T) method employed by Stanford biochemists to join pieces of DNA relied on the natural propensity of oligonucleotides, bits of single-stranded DNA, to hybridize with complementary sequences and form stable helical duplexes — the familiar double-chained, right-handed spiral structure of the molecule.
After Jim Watson and Francis Crick deduced the chemical composition and configuration of DNA in 1953, scientists around the world swarmed to decipher the genetic code and elucidate the mechanics of DNA transcription and translation. In the late 1960s and early 1970s, Stanford’s biochemists rode on the leading edge of this wave. They had learned enough about the properties of nucleic acids and enzymes to begin intervening in and manipulating the biochemical processes that constitute genetic replication.
Nucleic acids are chains of nucleotides. Nucleotides have three chemical components — a phosphate, a sugar, and a nitrogen-containing base (a purine, with a double-ringed chemical structure, or a pyrimidine, with single-ringed chemical structure). The chains are held together by covalent bonds between the phosphates and sugars of adjacent nucleotides.
Nucleotide chains are configured as stable double helices when hydrogen bonds form between complementary purine and pyrimidine bases. The purines in DNA are adenine and guanine; the pyrimidines are cytosine and thymine. Adenine (A) pairs with thymine (T), and cytosine (C) with guanine (G).
Peter Lobban and David Jackson took advantage of the molecule’s natural base-pairing properties to facilitate recombination. They used the enzyme terminal transferase to add single-stranded chains of (A) nucleotides to one piece of DNA and single-stranded chains of (T) nucleotides to another. When the complimentary chains found each other and combined, two DNA molecules were made into one.
Two industrial laboratories were also on the recombination trail. In April of 1971, a journal called Biochemical and Biophysical Research Communications published a paper on work with bacteriophage T7 by three researchers at the International Minerals and Chemical Corporation Growth Sciences Center in the Chicago suburb of Libertyville. The article was entitled, “Enzymatic Addition of Cohesive Ends to T7 DNA.” The project was ultimately unsuccessful — the T7 molecules resisted ligation.
The Illinois effort was unknown to the Stanford researchers, but Berg was aware of a proto-recombination project undertaken at Merck & Company by a former student of Jerry Hurwitz. Hurwitz and Berg had known each other for nearly twenty years. Hurwitz had earned a PhD from Western Reserve University a year after Berg, and he, too, joined Kornberg’s laboratory at Washington University in St. Louis.
In 1958, a year before Kornberg moved the group to Stanford, Hurwitz left to take a position at the New York University School of Medicine. Two years later, his lab was one of three that independently reported the discovery of RNA polymerase. In 1971, his former student at Merck began tinkering with the enzymatic manipulation of DNA molecules, although the practical utility of the work was not immediately apparent.
Merck let the project proceed for a number of months before shutting it down. The industrial scientists were at a distinct disadvantage — they didn’t have Kornberg’s refrigerator.
Closer to home, Bay Area academics outside Berg’s immediate circle were simultaneously closing in on the goal. Herb Boyer was working with restriction enzymes at UCSF, and thinking about ways to accomplish the in vitro recombination of DNA, but he still lacked the right combination of materials. Stan Cohen was at work in the Stanford University Department of Medicine, using plasmids to study the exchange of antibiotic resistance genes in bacteria. He monitored late developments in enzymology from a distance, but researchers in the field had not yet packaged methods enabling customized manipulations of plasmid DNA.
Berg’s lab was working in earnest on the development of such methods. By the fall of 1971, David Jackson had worked out reliable techniques for constructing complementary poly(A) and poly(T) tails on lambda dv gal and SV40 DNA. Lobban and Kaiser had already achieved success with the method and joined two pieces of P22 DNA — it was the first enzymatic recombination of discrete native DNA molecules. Earlier, Vittorio Sgaramella, J. Hans van de Sande, and famed DNA chemist Har Gobind Khorana had used bacteriophage T4 ligase to link double-stranded synthetic DNA molecules covalently.
Jackson’s progress had been spurred by a discovery made in Boyer’s UCSF lab. He had previously used an enzyme called DNase1 to open up SV40 for insertions. Under ordinary cellular conditions, the enzyme tended to sever just one of DNA’s twin strands, but in the presence of magnesium, it became invigorated and cut through both, so Jackson worked with it:
“There were all sorts of problems,” he says. “In addition to making these double-stranded cuts, we were putting all kinds of nicks in our DNA, the enzyme was making cuts in what appeared to be random locations, and the yield was low. I was making massive amounts of SV40 DNA, and purifying about 10 percent of it that contained full-length linear molecules — which is what we wanted — from the remaining super-coiled and relaxed circular DNA that was left. It was a nightmare.”
The problem was solved when Robert Yoshimori, one of Boyer’s postdocs, found a new restriction enzyme isolated from E. coli that cut DNA molecules at a specific recognition site, a specific nucleotide sequence. Fortuitously, the sequence appeared just once in lambda dv gal and once in SV40. Jackson used the enzyme, called EcoR1, to make one neat break in each, creating uniform linear molecules to which complementary tails could be attached. “Lo and behold,” he says, “the efficiency of the conversion of these SV40 circles into beautiful unit-length linear molecules was nearly 100 percent.” The Berg group was ready, for the first time in history, to connect by biochemical artifice pieces of DNA from different microorganisms.
The experiment had yet to be performed, but the line of inquiry had already stirred up trouble. The previous summer, Janet Mertz, a graduate student in Berg’s charge, had gone off to Cold Spring Harbor Laboratory to take a course on tumor viruses. Attendees were asked to say a few words about their interests and current research. Mertz described the recombination project in Berg’s laboratory.
She explained that, in theory, SV40 and lambda dv gal genes incorporated into recombinant molecules could be expressed in either bacterial or mammalian cells. Berg intended to transduce mammalian cells. Mertz also mentioned her additional plan to test the biological viability of recombinant genes in bacterial cells by putting SV40 DNA into E. coli. She hoped to replicate non-viable SV40 mutants in order to study their biological properties in monkey cells.
One of the course instructors, Robert Pollock, was alarmed by the idea. SV40 is a tumor virus. It causes tumors in rodents and induces oncogenic changes in human cells in culture. It is apparently harmless in monkeys, and perhaps in human beings, too. There is no evidence that SV40 causes disease in human beings. Still, for Pollock, the notion of inserting a known oncogenic virus into bacteria that inhabit the human intestinal tract instantly conjured up visions of a horrible plague — an infectious cancer.
Pollock called Berg to object to the experiment. Berg thought that the risks were minimal and that Pollock’s vehemence was misplaced. After the call, however, he felt compelled to canvass opinions from colleagues. He had long since come to terms with the risks attached to SV40, but understood Pollock’s reaction.
Initially, on arrival in San Diego, Berg had been leery of working with cancer viruses, but Dulbecco had assured him that SV40 was perfectly safe. According to a story told by virologist Shane Crotty in a biography of molecular biologist David Baltimore, Dulbecco swore the virus was so harmless he would drink it.
SV40
Simian virus 40 was first discovered in 1960 by legendary vaccine developer Maurice Hilleman at Merck, Sharp & Dohme, in the course of safety testing on Jonas Salk’s inactivated polio vaccine. It had evidently withstood the formaldehyde treatment used to inactivate the polio virus.
The potential pathogen was traced to the rhesus monkey and cynomolgus macaque kidney cells in which the vaccine was manufactured. Between 1955 and 1963, an estimated 98 million children and adults in the United States received doses of contaminated vaccine (government regulations issued in 1961 had required all vaccine preparations to be free of the virus, but previously manufactured vaccines stocks were not recalled).
Due to the scale of the public exposure, SV40 became the subject of intense biomedical scrutiny. Clinical and epidemiological studies have not produced consistent evidence associating SV40 with disease in human beings, but definitive statements on the question are precluded by the combination of established carcinogenic effects in rodents, in vitro evidence of cytopathic changes in human cells, and the theoretical possibility that sufficient genomic integration and cellular expression of viral oncogenes could generate tumors in human hosts.
The residual uncertainty is periodically exploited by journalistic accounts that weave tales of scientific irresponsibility and government cover-ups. For example, Debbie Bookchin and Jim Schumacher’s The Virus and the Vaccine, published by St. Martin’s Press in 2004, asserts that a large body of evidence implicating SV40 in growing incidences of lymphomas, mesotheliomas, and brain and bone cancers has been ignored or suppressed. Siding with scientific and medical dissenters, the authors attribute policy inaction not to insufficient data or a lack of consensus in the scientific community, but to biases reflecting political, commercial, and professional interests.
Berg was persuaded, but when he returned to Stanford with plans to do more research on viruses, he built an expensive P3 containment lab with filtered air, laminar flow hoods, and negative room pressure to safeguard against the unintended release of microorganisms, and to mitigate the concerns of employees working in and around the space. Some of the scientists with whom Berg consulted advised caution. Joshua Lederberg stressed the investigator’s responsibility, regardless of the outcome. “At that point,” Berg says, “I stepped back and asked, ‘Do I want to go ahead with experiments that could have catastrophic consequences?’”
In the fall of 1971, David Jackson created the world’s first recombinant DNA molecule in vitro. Berg postponed expression experiments and contacted Robert Pollock about organizing a conference on biohazard risks in research involving viruses.
Had Berg attempted expression with lamba dv gal, it would not have worked. It was later learned that EcoR1 disrupted a sequence necessary for replication of the plasmid. Mertz, however, prepared a modified version of lambda dv gal that she claims would have served as a viable cloning vector. She remembers discussing the work on a return trip to Cold Spring Harbor in August 1972. When asked about the status of the recombinant molecules, she replied, “I autoclaved them.”
The biohazards meeting was held in January 1973 at Asilomar State Beach, near Monterey. The Asilomar Conference Center was a regular site for Stanford retreats. Experts on tumor viruses were invited to discuss the perils of research in the field. The participants produced a lengthy report containing risk assessments and safety recommendations for the protection of laboratory workers.
One of the proposals was regular blood testing to detect antibodies indicating viral infection. Berg remembers: “We initiated a prospective study to collect blood samples. We still have them, frozen away. The purpose was to keep track of anybody who developed cancer and to check their prior exposures.” Berg asked his laboratory staff to get tested for antibodies against SV40. He learned that everyone in the lab was seroconverted — all had been exposed. Nobody got sick.
HERBERT BOYER
Herb Boyer hails from Derry, Pennsylvania, a small town in the Western part of the state, near Pittsburgh. His father was a brakeman and conductor for the Pennsylvania Railroad. As he grew into adolescence during the postwar era, Boyer’s life revolved around sports, hunting and fishing, and odd jobs.
He attributes his interest in science to a high school mentor, his coach in football and basketball who also taught algebra, trigonometry, plane geometry, solid geometry, biology, and chemistry. Boyer did well in school and became the first member of his family to attend college. In the fall of 1954, he matriculated at Saint Vincent College, a Benedictine school in nearby Latrobe, Pennsylvania.
To save money, he lived at home and commuted to classes. During summer recesses, he worked in a clinical laboratory at a local mental institution. He majored in biology and chemistry, and became fascinated with the recently resolved structure of DNA and its role in genetics.
Boyer went on to graduate work at the University of Pittsburgh, where he specialized in microbial genetics. It was an exciting time in molecular biology — efforts to crack the genetic code were underway, and the mechanisms that control gene expression were being explored in several different experimental models. Boyer worked with professor Ellis Englesberg on the genetic control of fermentation in E. coli. He examined the regulation of the L-arabinose operon, a cluster of genes that code for enzymes involved in the metabolism of arabinose, a pentose sugar.
The graduate apprentice used chemicals to induce mutations in these genes. He established that a mutation in the final step of the pathway leads to the accumulation of a salt, L-ribulose-s-phosphate, which inhibits cell growth. The finding provided a means of selecting for other mutants in the pathway. Boyer exposed the growth-retarded cells to mutagens once more, and plated them onto an arabinose medium (in which the genes would be activated). Cell growth indicated that secondary mutations had appeared earlier in the pathway. At that time, isolating mutants was difficult and time-consuming. Boyer’s method for direct selection of altered genes was a boon to Englesberg’s work on the arabinose operon.
The availability of mutants led to the formulation of Boyer’s dissertation research project. On the recommendation of Roger Weinberg, an assistant professor in the department, he attempted to generate data for deciphering the genetic code. The plan was to map genes by transduction of E. coli cells with bacteriophage P1. Boyer intended to correlate base pair changes in mutants with amino acid substitutions in expression products, a process that would enable him to make biological sense of gene sequences.
Boyer soon found that recombination frequencies in the P1 transduction system were very low. He had the idea that improved rates could be achieved through bacterial conjugation. All of Boyer’s work on the L-arabinose operon had been done in the B strain of E. coli, but high frequency recombination systems had been developed in E. coli K. Boyer decided to cross the strains. The switch in methods led him in a roundabout and unexpected way to the study of restriction enzymes.
In the course of the mapping work, Boyer made a puzzling empirical observation. He compared rates of genetic recombination in inter- and intra-strain bacterial conjugations and found that they were significantly lower in the former. It appeared that E. coli B was somehow recognizing non-homologous (foreign) DNA from E. coli K and interfering with recombination.
Boyer could not explain the anomalous result. He was disappointed because he hadn’t solved the recombination frequency problem, but he soon found that the phenomenon was tied to a set of genes located near the arabinose operon. When select alleles of these genes were transferred from E. coli K to E. coli B, the effect disappeared. Although he could not yet precisely describe the control mechanisms, Boyer had discovered the genes that govern restriction and modification systems in the two strains.
In the middle of the mapping project, Boyer’s grand goal of cracking the code was accomplished by others through methods of in vitro polypeptide synthesis. The original pretext for his dissertation research had evaporated, but Boyer was already thoroughly engrossed in a new line of inquiry — the study of cellular systems that control the selective recognition and processing of DNA.
He subsequently learned that Werner Arber, a professor at the University of Geneva, and Daisy Dussoix, a graduate student, had one year earlier hypothesized the presence of enzymes that work in tandem to perform the tasks of restriction and modification — endonucleases that destroy foreign genes by cutting up DNA molecules at or near specific DNA sequences that serve as recognition sites, and methylases that protect identical sequences in host DNA from recognition and restriction.
When Boyer moved next into a post-doctoral position in microbiology at Yale, in the lab of Ed Adelberg, he took on the project of isolating the enzymes presumed to be responsible for restriction and modification in E. coli B. Research in the area was just beginning, much was unknown, and Boyer selected endonucleases and methylases that he later learned were peculiar and structurally complex. He failed to recognize that their biological activity depended on the presence of certain chemical cofactors, which prevented him from isolating and characterizing the enzymes.
When his stay at Yale was drawing to a close, Boyer went to his advisor for counsel and assistance. Adelberg had recently written a microbiology textbook for medical students with Ernie Jawetz, Chair of the Department of Microbiology at the University of California, San Francisco (UCSF). He had heard from Jawetz that a position was opening up at UCSF, and that the university administration was committed to strengthening scientific research at the school.
UCSF was known as a provincial teaching institution, but had decided to upgrade its basic biological and biomedical science departments. Adelberg recommended the opportunity. According to Boyer, he said, “You should look into it. It should develop into a very good center for the basic sciences.” Boyer did look into it, and was offered a job. He started as an assistant professor of microbiology in the summer of 1966.
Herb and his wife, Grace, liked the idea of moving to the West Coast. “I had always wanted to come to California,” he says. “I thought it was great, very different than the East.” Boyer was excited by his first faculty appointment and his new independence. He was delivered from scut work, tenement apartments, and dreary winters — the lot of graduate students and scientific apprentices in Pittsburgh and New Haven.
By comparison, San Francisco was enchanting. The UCSF campus on Parnassus Heights overlooked Golden Gate Park, the city’s lively Inner Sunset and Haight-Ashbury districts, and, beyond, the headlands of Marin County. The views were expansive, breathtaking. Boyer loved all of that, but his high spirits were deflated when he considered his cramped work space.
He had been promised a laboratory in one of the university’s two newly constructed health sciences towers, on a floor occupied by the rest of the microbiology department, but was relegated instead to three tiny rooms in the old Medical Sciences Building. The official rationale was that tissue culture work was being done in the towers, and it wouldn’t do to have bacteria in the vicinity.
Boyer’s antiquated facilities were designed for work in classical microbiology, for specimen plating, slide staining, and microscopy. They were not well-suited to the separation and fractionation techniques employed in molecular biology. The laboratory lacked pieces of essential equipment. Boyer can still recite the litany of problems with his space:
“There was an autoclave nearby, but no ultracentrifuge. In order to do any enzyme purification, we had to walk over to Health Sciences East. The people there graciously let us in, but they weren’t exactly thrilled to see us.” Boyer’s lab also lacked a cold room. He didn’t even have an ice machine: “Every morning, we had to go over to Health Sciences West to get large buckets of ice in which to store our specimens. It was extremely difficult to carry out research. Eventually, I got some funding to install a cold room. We lost laboratory space, but we needed to do it.”
Boyer also dreaded giving lectures to medical students, who generally had little interest in bacterial genetics: “It put the fear of God into me to face that group. I didn’t know how to make it interesting.” Jawetz, the department chair, would occasionally sit in on his classes to make performance assessments. Boyer was mortified: “I thought, ‘Oh my God, oh no.’ It was hard on the ego to be told, ‘You’d better get it together here.’”
In addition to feeling pedagogically inadequate, Boyer felt scientifically isolated. Aside from virologists Leon Levintow and Warren Levinson, no one in the microbiology department worked in related areas of inquiry, and no one appeared to have much interest in or knowledge of his specialty. Interactions between Boyer’s lab and others were limited. There were no collaborations. His colleagues were civil, but Boyer perceived an absence of camaraderie.
When he could, he traveled across the Bay to the University of California, Berkeley, or down the peninsula to Stanford, to attend seminars and converse with like-minded scientists. To Boyer, it seemed that molecular genetics was moving ahead at these places while UCSF was standing still. It all added up to a sense of professional discontent. “Within a year,” he says, “I was looking at other positions. I was very dissatisfied.”
As it happens, he didn’t manage an exit, and the environment slowly started to improve, along with his outlook. Virologist Michael Bishop arrived as a new faculty member in 1968, and provided “scientific stimulation” and “good company.” The following year, Bill Rutter agreed, after extended negotiations, to head the reorganized Department of Biochemistry and Biophysics.
Rutter moved UCSF directly onto the cutting-edge of molecular genetics. He recruited top scientists in the field, who in turn attracted the best graduate students. Between 1970 and 1973, Harvey Eisen, Howard Goodman, Christine Guthrie, Regis Kelly, Brian McCarthy, and Gordon Tomkins came into the department as new faculty members. After Rutter appeared on the scene, says Boyer, UCSF “was a much different place.”
Boyer felt a strong intellectual affinity with the biochemists’ research agendas. He began collaborating with Goodman and others. He saw that the atmosphere in Rutter’s world was highly competitive, but also highly collegial. His ties to biochemistry deepened, and later, when the opportunity arose to obtain new laboratory space in the department, he transferred in from microbiology.
As the quality and reputation of UCSF biochemistry began to improve in the early 1970s, so did Boyer’s scientific prospects. He had continued to chase restriction and modification enzymes since arriving in California, without much luck. In 1970, Hamilton Smith of the Johns Hopkins University School of Medicine reported the first isolation of a site-specific restriction enzyme, a molecule found in Hemophilus influenzae called HindII, along with the nucleotide sequence of the recognition site.
It was the Holy Grail in Boyer’s chosen corner of the scientific world. Smith was later awarded a Nobel Prize for finding it. Boyer recalls his reaction to the news: “So frustrating!” In the spring of 1971, the wheel of fortune began to turn. Robert Yoshimori was working on a doctoral thesis under Boyer’s tutelage when he retrieved a new restriction endonuclease from a clinical sample of E. coli. The sample had been taken from a female patient at the UCSF Medical Center with a urinary tract infection that displayed resistance to multiple antibiotics.
It was EcoR1, the miracle enzyme. Boyer investigated its properties, and found that it consistently cut lambda phage DNA into a unique set of fragments. The news traveled fast. Boyer began supplying the enzyme to others in the field on request. Paul Berg received a sample, and distributed it to Stanford colleagues to see if it had use in investigations of the SV40 genome. The availability of EcoR1 triggered a rapid succession of consequential discoveries.
David Jackson acquired the enzyme and began using it to cut SV40 and lambda dv gal. In the spring of 1972, three researchers at the National Institute of Allergy and Infectious Disease (NIAID) in Bethesda, Maryland — George Fareed, Claude Garon, and Norman Salzman — determined that EcoR1 (supplied by Boyer) cleaved SV40 DNA reliably at a single specific site. Berg’s graduate student John Morrow made the same observation independently, and then provided EcoR1-generated SV40 restriction fragments to Vittorio Sgaramella, who was visiting Joshua Lederberg’s laboratory in the Stanford University Department of Genetics.
Sgaramella assumed that the SV40 DNA was blunt-ended. He tried to join it to pieces of blunt-ended P22 DNA with a ligating enzyme isolated from the bacteriophage T4. He found that the ligase joined pieces of P22 together, and pieces of SV40 as well, but it did not combine P22 and SV40 DNA. He concluded that the recombination likely failed because EcoR1 left single-stranded cohesive ends on the SV40 fragments, and these could not be joined with the blunt duplexed ends of P22 DNA.
In the meantime, Janet Mertz, Berg’s graduate student, had also acquired EcoR1 for experimentation with SV40. Her inquiries established securely that the enzyme produces cohesive ends when it cuts DNA molecules — single-stranded tails primed for hybridization. Mertz had expected cleaved rings of SV40 DNA to behave like linear molecules, but they didn’t — they were infectious in monkey cells. She examined the DNA by electron microscope and saw that the restricted molecules had reformed as rings. The complementary cohesive ends had re-annealed.
Two days later, Berg’s faculty colleague Ronald Davis examined EcoRI-cut SV40 DNA that had been mounted on a microscope in the cold room. The restriction fragments had not been incubated with DNA ligase, but Davis also saw circles. At low temperatures, ligase wasn’t necessary for the cohesive ends to re-anneal or hybridize.
Mertz traveled up to San Francisco with John Morrow to inform Boyer of the finding. Boyer had been working with UCSF colleagues Joe Hedgpeth and Howard Goodman to sequence the nucleotides that compose the EcoR1 recognition site. They had assumed that the enzyme made a blunt-ended cut. With the information provided by Mertz and Davis, Boyer went back to the lab, finished sequencing the recognition site, determined the enzyme’s distinctive cleaving pattern, and characterized the resulting complementary termini.
Word soon got out about the restriction enzyme that made ‘sticky ends.’ EcoR1’s specificity and its peculiar slicing action made it a powerful tool for the manipulation of DNA. Researchers suddenly possessed the means to prepare, with relative ease, an unlimited set of recombinant molecules.
Sticky Ends
Prior to the discovery of EcoR1, the recombination of genetic material was, as Paul Berg has conceded, “cumbersome, technically challenging, and perhaps not replicable.” EcoR1 changed all of that.
EcoR1 is a restriction endonuclease, an enzyme that cuts DNA molecules into pieces. Scientists believe that restriction enzymes evolved in bacteria to defend against infiltrating viral genomes. The first molecular specimens were identified in the late 1960s. Biochemists undertook a broad search to find others. No one knew how many awaited discovery. EcoR1 turned up in San Francisco in 1971, and it was a boon to DNA recombination projects.
As a type II restrictor, EcoR1 reliably severs DNA at a specific recognition site, a definite DNA sequence, and always makes the same precise cut within it. As Boyer learned on a Saturday morning in the spring of 1972, EcoR1’s recognition sequence is:
GAATTC
CTTAAGThe enzyme cleaves between the G and A nucleotides on both strands to produce a signature staggered cut:
G / AATTC
CTTAA / GBoth pieces of the severed molecule are left with an overhang of four nucleotides at one end. The sequences are palindromic complements: AATT and TTAA. Mertz and Davis called the ends ‘sticky’ because, in contrast to restricted DNA molecules with blunt ends, the overhangs on the EcoR1-restricted fragments will readily combine with complementary sequences. And because the enzyme makes identical cuts in any piece of DNA from any organism, fragments produced by EcoR1 restriction are naturally complimentary, and can be joined without further biochemical treatment.
EcoR1’s ‘sticky ends’ permitted researchers to cut and paste DNA with far greater ease, flexibility, and precision than prior methods and materials. It served as an important enabling tool in the development of recombinant DNA technology.
Stan Cohen heard about the enzyme from a colleague in San Diego, molecular biologist Don Helinski. Cohen and Helinski were organizing an international conference on plasmids to be held in November, in Hawaii. Helinski suggested that Cohen extend an invitation to Boyer to talk about the significance of EcoR1 in plasmid genetics.
Stanford and UCSF authors simultaneously published four papers on EcoR1 in the November 1972 issue of the Proceedings of the National Academy of Sciences (PNAS): Morrow and Berg, Mertz and Davis, Sgaramella, and Hedgpeth, Goodman, and Boyer.
STANLEY COHEN
Stan Cohen was born and raised in Perth Amboy, New Jersey. His father ran small retail businesses that sold light fixtures and electrical supplies, but didn’t do particularly well. Cohen’s mother worked as a secretary. Both parents encouraged hard work and academic accomplishment, and Cohen was an eager student. He enjoyed writing and developed broad interests in the sciences.
In the mid-1950s, Cohen earned a scholarship to Rutgers University, which enabled him to stay close to home and near his father, whose health was failing at the time. He had decided on medicine as a future career. He followed a pre-med course of study while engaging in numerous extracurricular activities, including the debating club.
He also pursued an interest in music. Cohen wrote and sold a song called ‘Only You’ that was recorded by Billy Eckstine & The Pied Pipers and made it onto the ‘Hit Parade’ in 1955. Modest royalty payments from the tune helped him to finance his education.
From Rutgers, Cohen went to the University of Pennsylvania School of Medicine. He received financial assistance from the university and from the Robert Wood Johnson Foundation. Most of his educational expenses were covered. He enjoyed medical training and living in Philadelphia. During his second year, Cohen initiated what soon became an occupational habit — creating opportunities to conduct interesting research by making connections with leading investigators in the field of interest.
Cohen had become fascinated by reports of immunological rejections of transplanted tumors, and arranged to work in a pathology lab. He undertook an immunological study of cross-species skin grafts in hamsters and rabbits, and enlisted the aid of Rupert Billingham, an expert in transplantation immunity at Philadelphia’s Wistar Institute.
Billingham had trained in London with Peter Medawar, one of the world’s foremost authorities in immunology. Through Billingham, Cohen finagled an extended stay in Medawar’s laboratory, from May through September, 1959. Before returning to the United States, he traveled around the British Isles and Europe, with funds raised by playing the banjo and singing in pubs and cafes. Back in Philadelphia a few months later, he learned that Medawar had been awarded a Nobel Prize for the discovery of acquired immune intolerance.
Cohen had planned an internship and residency in internal medicine, but decided instead to follow a career path into academic research. He finished medical school, and served from 1962 through 1964 as a clinical associate at the National Institute of Arthritis and Metabolic Diseases (NIAMD) in Bethesda, Maryland. He remembers the institute as “an idyllic environment” in which to work.
It was during this period that Cohen was introduced to DNA research. He was able to consult with top investigators in the field simply by walking down the hall. He drew a fateful conclusion from the experience: “I needed to know a lot more about biochemistry and genetics.”
After resolving to complete his clinical training, and taking a year to serve as a resident in internal medicine at the Duke University Hospital, Cohen moved into an American Cancer Society post-doctoral fellowship under Jerry Hurwitz, the defector from Arthur Kornberg’s Washington University group, at the Albert Einstein College of Medicine in the Bronx.
Most of the work in Hurwitz’ lab was focused on the chemistry of functional interactions between enzymes and nucleic acids, but Cohen was assigned a project in lambda phage genetics. Viral replication occurs within infected cells in stages. Cohen was to look for evidence of selectivity in the transcription and activation of ‘early’ and ‘late’ stage viral genes by bacterial RNA polymerase.
The work served as Cohen’s introduction to fast-track molecular biology. He generated some publishable results, and molecular biologist Mark Ptashne, who was likewise working on gene regulation in lambda phage at Harvard at the time, took note. Ptashne invited the young physician to give a seminar talk in Cambridge. Jim Watson attended.
Cohen reports that Watson “sat in the first row and read the New York Times.” The behavior was disconcerting: “Presenting my work in that setting as a postdoc was a big event for me. I was depressed that Watson found it boring.” Cohen felt better when, at the end of the session, Watson asked a number of insightful questions: “it was clear he had been listening.”
Cohen spent the summer of 1966 in courses on bacterial and phage genetics at Cold Spring Harbor Laboratory. One of the guest lecturers, Richard Novick of the Public Health Research Institute in New York City, addressed the relationship between antibiotic resistance in bacteria and plasmid DNA. Little was known at the time about the genetics of antibiotic resistance, but there was some evidence implicating exchanges of plasmid DNA in conjugation and transformation.
Cohen understood the medical significance of the outstanding questions — bacterial resistance to antibiotics was becoming recognized as a serious public health issue. Cohen recalled the story of a medical resident at the University of Pennsylvania who had died after contracting an untreatable staph infection. He decided that he would take up research on the genetics of bacterial antibiotic resistance.
Plasmid research was attractive to Cohen, in part because it remained “a backwater of molecular biology.” At the time, phage genetics was the hottest area of inquiry in the field. Competition within it was fierce. Cohen was afraid that he would not be able to keep up.
He was wrestling with a dilemma. His fellowship was winding down, and he was gearing up to conduct a search for a permanent faculty position. Cohen envisioned taking a job in a clinical department: “I decided, in the end, that I had invested so much of my life being trained in clinical medicine that I would try to combine clinical activities with basic research.”
Medical schools were encouraging clinically engaged faculty to conduct fundamental inquiries in biomedical science, on the assumption that innovative approaches and tools would be imported to medical practice from the sciences. Moreover, in the late 1960s, ample funds were available from the National Institutes of Health (NIH) for research in clinical settings.
Still, Hurwitz tried to discourage Cohen’s impulse to keep a hand in doctoring. He believed it would be difficult for Cohen to maintain a serious research program with clinical responsibilities depleting his time and energy. Cohen thought that he could perhaps pull it off in a relatively slow-moving but medically relevant area such as plasmid genetics.
Plasmids
Plasmids, circular ring-shaped molecules that carry extrachromosomal DNA, were first identified and named by Joshua Lederberg in 1952. Subsequent research determined that plasmids replicate autonomously. Their purpose is to transfer genes horizontally across populations, usually through bacterial conjugation, a process in which a donor cell passes a mobile piece of DNA, such as a plasmid, to an adjacent cell during direct physical contact. Plasmid DNA typically encodes instructions for making proteins that afford adaptive advantages — antibiotic resistance, for example, or the ability to break down organic compounds into nutrients. Plasmids do not carry genes that code for proteins involved in essential cell functions. These are conserved on chromosomes.
In 1967, Cohen attended a conference on antibiotic resistant plasmids at Georgetown University. The meeting had been organized by microbiologist Stanley Falkow, a leading pioneer in the emerging field (only in 1962 had Tsutumo Watanabe discovered that resistance could be transferred via plasmids). Cohen approached Falkow after a conference session and explained his intent to enter the territory.
Falkow was encouraging and helpful. He agreed to send plasmid strains that Cohen would need to establish an experimental program. Cohen began surveying the field in earnest while contemplating offers to serve on the medical faculties of the University of Pennsylvania and Duke University. In the end, he decided that neither was a good fit.
Hurwitz arranged a one-year extension to keep Cohen at Albert Einstein while he explored other options. As the year progressed, Hurwitz conversed with old friends from Washington University, Dale Kaiser and Paul Berg, both now in Stanford’s Department of Biochemistry, about the physician-immunologist-plasmid geneticist housed in his laboratory. Berg knew about Cohen’s work on phage and RNA polymerase, and volunteered to make inquiries with Halstead Holman, Chair of the Stanford Department of Medicine. Holman, Berg knew, was a strong proponent of the hybrid clinician/basic researcher role. Cohen fit the bill.
Interviews and negotiations ensued. Cohen was offered a position as an assistant professor in the Stanford Department of Medicine — but within the Division of Hematology. Cohen hadn’t been trained in hematology, but said he was willing to give it a try. When he arrived at Stanford, his lab space was occupied, on temporary loan to others, but he was offered temporary lodging in biochemistry.
Cohen established close ties with many younger researchers in the department, including post-docs and graduate students. He participated in seminars, shared research materials, and gave and received advice and assistance. For a time, he hoped to secure a joint appointment in clinical medicine and biochemistry, but was disabused of the notion when he went to see Arthur Kornberg, the chairman of the biochemistry department.
Kornberg was also a physician. He had given up clinical practice long ago to concentrate on research. He didn’t think the two could be effectively combined: “If you’re going to be seriously involved in research,” he told Cohen, “then you’re going to neglect your clinical responsibilities.” Kornberg had assembled an elite department. He wasn’t about to compromise its reputation for excellence by welcoming half-time dilettantes. He added that he considered plasmids “not very interesting.”
After trying for a year to take to hematology, Cohen decided that it probably wasn’t going to happen. He revealed his dissatisfaction to Hal Holman, who wanted to keep Cohen on the staff and graciously offered to entertain an alternative arrangement. Cohen had become interested in the biochemistry of drug interactions, so he suggested starting a Division of Clinical Pharmacology. Holman gave his assent.
For a time, Cohen was the only faculty member in the Division. His first project was a computer-based reporting system to warn physicians of possible adverse interaction effects. Eventually the unit grew and became successful. Cohen directed it until 1978, when he left to become the Chairman of Stanford’s Department of Genetics.
While starting the new division, Cohen moved ahead with the plasmid research. To investigate the structure and function of genes conferring antibiotic resistance, he needed a method for introducing plasmid DNA into E. coli cells. It hadn’t been done before. No one had been able to effect artificial genetic changes in E. coli by means of transformation.
A critical break occurred in 1970, when biochemists Morton Mandel and Akiko Higa of the University of Hawaii published a letter in the Journal of Molecular Biology reporting that the uptake of DNA by E. coli is dependent on the presence of calcium ions. They had managed to transform an E. coli cell with a virus, lambda phage.
Cohen decided to try it with plasmids, to see if calcium would facilitate transformation with extrachromosomal bacterial DNA. He assigned the project to Leslie Hsu, a medical student working in his laboratory. Hsu treated a non-resistant strain of E. coli with calcium chloride, and introduced plasmids carrying genes for antibiotic resistance. She transferred the cells to a culture medium containing antibiotics and was able grow resistant colonies. The plasmid DNA had been taken up and propagated. The resistance genes had been expressed.
Cohen published the result in PNAS in August of 1972. The paper did not make a broad impact in molecular biology. The relatively small community of plasmid researchers was very pleased to learn that E. coli, once refractory to transformation, could now be coaxed into it. That opened a window on a wide range of experimental possibilities. But Cohen felt that molecular biologists had neglected the most important implication of the work.
The experiment had demonstrated a method of cloning cells that contained autonomously replicating DNA molecules. Previously, only phage DNA could be cloned in this way. Now, there was a method available for cloning entire extrachromosomal bacterial genomes. The lack of a broader realization was fine with Cohen: “It gave me time to proceed further without the pressure of intense scientific competition.”
THE COHEN-BOYER COLLABORATION
In November 1972, the plasmid conference was held in Honolulu. It brought together researchers from the United States and Japan, a country in which prescribing multiple antibiotics to combat infections had become a common medical practice. As a result, the problem of bacterial strains exhibiting multiple resistances had become especially acute there, and many Japanese biomedical researchers had been drawn to plasmid studies.
Boyer had been invited to the meeting to talk about EcoR1. He met Cohen, who described his work. Boyer offered to send EcoR1, and they began to brainstorm about possible experiments. On an evening with time off from conference proceedings, Boyer and Cohen met up with mutual acquaintances Stan Falkow and University of Pittsburgh microbiologist Charles Brinton, with whom Cohen was collaborating, and Brinton’s wife, Ginger.
The group took a stroll on the streets running along Waikiki beach. They stopped at a delicatessen, where topics in plasmid genetics were discussed over sandwiches and beer. The talk centered on the new horizon of experiments that EcoR1 had brought into view. Cohen and Boyer sketched a possible method for cloning genes in E. coli. By the end of the meal, they had reached a solid agreement to collaborate.
On his return to the mainland, Boyer was scheduled to give a talk at Cold Spring Harbor Laboratory. He was met at the airport in New York by molecular biologists Phillip Sharp and Joe Sambrook. They were excited to show him a new technique they had developed for differentiating and characterizing DNA molecules.
Boyer was ushered into a darkroom at Cold Spring Harbor and shown an agarose gel in which fragments of restricted adenovirus DNA had been separated by electrophoresis and stained with ethidium bromide, a dye that fluoresces intensely when it binds with nucleic acids. Boyer was astonished: “It was one of the most exciting things I could have looked at. I said, ‘Thank you, Lord!’” In an instant, he had a solution to the problems that had been impeding forward progress in recombination experiments he had started earlier in the year with Robert Helling, a visiting professor in his laboratory.
With this innovation, the separation of DNA molecules by size was immediately apparent on visual inspection. What had previously taken hours of tedious, uncomfortable work could now be accomplished in a matter of minutes. Boyer ran through the experimental steps in his mind, and concluded that, in relative terms, his research on DNA endonucleases would soon start moving like lightning: “When I saw those gels,” he says, “I couldn’t wait to get back home and get back to the laboratory.”
This Week’s Citation Classic
In 1988, Current Contents, a print compendium of bibliographic information on recently published studies in academic science, published a note by Robert Helling on what was then, according to the Science Citation Index, the most cited article in the Journal of Virology. The article, entitled “Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis,” had been published in November 1974 and subsequently cited in 605 scientific publications. The authors were Helling, Howard Goodman, and Herb Boyer. The paper reported on work conducted in 1972 and 1973 at the University of California, San Francisco.
Helling, a faculty member at the University of Michigan, was spending a sabbatical leave in Boyer’s laboratory. The two scientists, friends from graduate school at the University of Pittsburgh, had been attempting the in vitro recombination of lambda phage DNA with the plasmid lambda dv gal, but hadn’t managed it.
Helling and Boyer needed a better way to identify and characterize restriction fragments, the pieces of DNA they were attempting to join. With input from Goodman and Boyer, Helling developed one. The article described a method for separating DNA fragments by molecular weight and charge. Restriction enzymes chop DNA molecules into pieces. Helling’s separation techniques permitted the researchers to state with confidence what had become of the original molecules.
Previously, the standard technique for separating and analyzing DNA had been sucrose-gradient centrifugation, a cumbersome method with extremely low resolving power. Helling, Goodman, and Boyer’s alternative involved running DNA through multiple procedures — the innovative agarose gel electrophoresis method reported in the paper, along with radiolabeling and scintillation counting. It was awkward, messy, and time-consuming, but it yielded superior results.
Helling’s note credited Phil Sharp and Joe Sambrook’s ethidium bromide staining technique with greatly simplifying the procedure and eliminating the need to use radioactive materials. The staining method likewise became a citation classic, in the journal Biochemistry. The original report was published in 1973. By 1982, it had been cited 1,040 times. As these citation patterns indicate, both systems became widely adopted.
The Boyer-Cohen collaboration on recombinant DNA technology got underway in January. There were two clinching experiments. The work on each was performed at two sites. Boyer and Bob Helling worked at Boyer’s UCSF laboratory; Cohen and research technician Annie Chang worked in Cohen’s Stanford laboratory.
In the first experiment, the researchers isolated two plasmids, each from a different E. coli strain. The plasmids came from Cohen’s lab. They were called pSC101 (for ‘plasmid, Stanley Cohen, 101’) and pSC102. The first carried a gene for tetracycline resistance; the second contained a gene for resistance to kanamycin. Purification of the plasmid DNA was performed at Stanford. Annie Chang lived in San Francisco and commuted to Palo Alto. At the end of the day, on her way home, she transported the purified DNA to Boyer’s lab at UCSF.
Boyer and Helling used EcoR1 to cleave the plasmids into pieces, leaving linear DNA molecules with ‘sticky ends,’ primed for recombination. The fragments were mixed together and permitted to recombine naturally in various ways. DNA ligase was added to anneal the resulting bonds, and the molecules were analyzed by gel electrophoresis.
On her way to work the next morning, Annie Chang picked up the processed DNA at UCSF and carried it back down the peninsula to Stanford. There, the plasmids were examined and characterized by electron microscopy (a technique Cohen picked up from a collaboration with Norman Davidson and Phil Sharp at Caltech) and mixed with E. coli in a suspension of cold calcium chloride. The temperature was rapidly raised and lowered, creating a heat shock that induced the bacteria to take up the DNA. The transformed bacteria were spread on a plate containing tetracycline and kanamycin. Only bacteria resistant to both could survive.
It was possible that bacteria exhibiting dual resistance could contain both pSC101 and pSC102 that had been reassembled by DNA ligase, so the plasmids were retrieved and subjected once more to restriction by EcoR1. Separation of the fragments by gel electrophoresis produced evidence that artificially engineered recombinant plasmid DNA had been cloned and expressed for the first time in history. Boyer recalls examining the critical gels with Bob Helling: “I can remember tears coming into my eyes. It was so nice.”
Boyer attended the Gordon Conference on Nucleic Acids in June and let the news out. Says Cohen: “I wasn’t very happy about it because Herb and I had agreed not to talk. On the other hand, I realized that it’s difficult to avoid telling others about results that are so exciting. When you have an exciting finding, you want to let colleagues know about it.” Boyer’s recollections are different. He believes that Cohen knew he would discuss the experiment.
In any case, Boyer’s comments became the focal point of the conference, and sparked discussion and debate about potential biohazards. The co-chairs of the conference, Maxine Singer and Dieter Söll, called a special session to address the issue. After an open airing of views, two votes were taken. By an overwhelming majority, the group authorized a request to the National Academy of Sciences for the formation of a scientific panel to study the public health and safety implications of genetic engineering, and to consider the ethical dimensions of risk-laden experimentation.
A second referendum revealed fractures and conflicts in the community of researchers. Singer and Söll proposed publishing a letter in Science (that they had already drafted) to express the sentiments and opinions of scientists on the matter. The motion passed, but by the narrowest margin. Many researchers felt that a published statement on biohazards would provoke unwarranted public fears. It didn’t. The letter appeared, but garnered scarce attention.
An important seed for the second Boyer-Cohen experiment was sown at the same conference. Boyer recalls a conversation with a graduate student from Paul Berg’s lab, John Morrow: “I said, ‘The next thing we have to do is recombine a piece of eukaryotic DNA to the plasmid and show that it can replicate. We’re looking for the right sample.’ And John said, ‘I have some amplified ribosomal DNA from Xenopus laevis [an amphibian, an African frog].’” Morrow had acquired the DNA from Don Brown at the Carnegie Institution for Science in Baltimore, and found that it was neatly cleaved by EcoR1. Boyer and Morrow discussed a collaboration.
In 1973, very few eukaryotic genes had been purified and characterized. The available methods were still primitive. Morrow was offering a rare commodity, a valuable gift. At the time, Berg had temporarily suspended his research on recombinant DNA, and knew nothing of Morrow’s involvement: “Brown had sent him some frog ribosomal DNA, for what reason I don’t know. It was in Morrow’s refrigerator. Unbeknownst to me, he went to Cohen and Boyer and said, ‘We ought to be able to introduce this frog DNA into E. coli by linking it to the plasmid.’ Eventually, the experiment was done, and John came to me and told me about it. I almost kicked him out of the lab, I was so furious.”
When Boyer returned to his office, he telephoned Cohen to tell of the acquisition, and to invite his participation in the next experimental round. There remained a good deal of uncertainty surrounding the project. Boyer and Cohen had run into few problems in cloning antibiotic resistance markers in E. coli, and Cohen and Chang had subsequently performed the first interspecies transfer and expression of bacterial DNA when they spliced an antibiotic resistance gene from a gram negative Staphylococcus aureus bacterium into a plasmid, transformed gram positive E. coli cells, and recorded effective reproduction of the gene and transmission of the trait. Still, no one knew whether it was possible to propagate and express eukaryotic genes in simple prokaryotic systems.
Even if replication and expression were to be achieved, it was not immediately clear how the DNA could be recovered. The first experiment involved genes for antibiotic resistance, the presence of which could be readily confirmed by exposing bacteria to drugs. There were no such methods available for selectively monitoring the uptake and propagation of eukaryotic genes. If the cloning process was inefficient, fishing eukaryotic genes out of bacterial cells could be difficult and time-consuming.
They went ahead with the work. Following the general model of the first experiment, the scientists cut pSC101 and ribosomal DNA with EcoR1, mixed the fragments, and treated newly recombined molecules with DNA ligase. They were able to transform the bacteria and select for tetracycline resistance. They extracted plasmids from surviving cells, purified them, analyzed their physical conformations by electron microscopy, and restricted them once more. The frequency of recombination was surprisingly high. The recut DNA was characterized by gel electrophoresis, and compared with earlier runs of restricted psSC101 and eukaryotic DNA. The pieces of the puzzle fit together.
Cohen suggested that further supporting evidence for the identity and presence of amphibian ribosomal DNA could be gathered through centrifugation of restriction fragments and analyses of differences in buoyant density. The buoyant density of DNA is a function of G-C (guanine and cytosine) content. Eukaryotic and prokaryotic DNA differ in degrees of G-C richness. The centrifugation of recombinant plasmid fragments showed that suspected Xenopus genes displayed the expected buoyant density as well as the expected size.
Cohen and Boyer also found that Xenopus genes were expressed. They knew that amphibian ribosomal RNA had been manufactured in the bacteria because they found it hybridized to the amphibian DNA. “That was a little bit of icing on the cake,” says Boyer, “that confirmed that we had cloned eukaryotic ribosomal DNA.” The second experiment was a resounding success. Cohen remembers that “the work proceeded quickly. It was an extremely exciting time. It was a continual high.”
The experiments constituted a breakthrough in many ways. Boyer comments on the scientific significance of the demonstration, and the breadth of its technological impact: “A lot of my mentors and colleagues were leaving microbial systems to study higher order cells because ‘everything there was to know about bacteria was known.’ But they were frustrated because they had no hope to isolate single genes or fragments of genes from the chromosomes of ‘higher organisms.’ When I looked at those gels, I knew we’d be able to isolate any piece of DNA that was cut with EcoR1, regardless of where it came from.”
The invention of recombinant DNA technology opened a gateway to scientific and technological possibilities that just a few years earlier would have been dismissed as fantastic imaginings, flights of fancy. Cohen and Boyer had hinted at the general utility of recombinant DNA technology in the closing paragraph of the November 1973 paper that reported the results of the first experiment in PNAS. They wrote: “The procedure described here is potentially useful for insertion of specific sequences from prokaryotic or eukaryotic chromosomes or extrachromosomal DNA into independently replicating bacterial plasmids.” The second experiment confirmed the utility of the approach.
There were also practical implications to consider — applications in medicine, agriculture, and the chemical industry. Mary Betlach, a technician in Boyer’s laboratory who made significant contributions to both experiments, remembers that her boss had been thinking about the practical value of the work from the time she joined the lab in 1972: “Herb was talking early on about cloning insulin. I used to think, ‘That’s a little far-fetched.’ But he kept saying, ‘We can probably clone insulin,’ or ‘This could benefit society.’”
CONTROVERSIES
The publication of the Xenopus DNA results concluded the Cohen-Boyer collaboration, but not the entanglement of the two scientists in related controversies. By pursuing recombinant DNA, Cohen and Boyer had arrived at a crucial turning point in the development of molecular genetics, and at the center of legal and ethical controversies that would carry on for another decade. Their seminal experiments had provided life scientists with a technology for isolating, purifying, and manipulating any genes from any organism, and perhaps expressing them as well, but paths of further research were clouded in uncertainty. News of the breakthrough and commentaries by scientists and other observers on possible risks and benefits had engendered in the public mind both hopes and fears.
In April 1974, a month before the publication of the second Cohen-Boyer paper, Berg convened a group of prominent molecular biologists to consider a request for policy advice from Phillip Handler, President of the National Academy of Sciences (NAS). David Baltimore, Dan Nathans, Richard Roblin, Jim Watson, Sherman Weissman, and Norton Zinder met with Berg at MIT. The group, which was soon expanded to include Herb Boyer, Stan Cohen, Ronald Davis, and David Hogness, deliberated and recommended to Handler and the NAS a policy of caution, even though most were of the opinion that risks were negligible.
On May 20, the New York Times published an article on the paper by Victor McElheny, entitled “Animal Gene Shifted to Bacteria.” The next month, Newsweek magazine picked up the thread with a story on “The Gene Transplanters.” The piece ran through the list of benefits to be realized in the future through the wonders of genetic engineering — medical miracles, agricultural bounties, industrial transformations.
Back in Palo Alto, Niels Reimers, director of the Stanford Office of Technology Licensing read the New York Times piece, and immediately got Cohen on the phone: “Stan,” he said, “this looks very interesting.” According to Reimers, Cohen replied, “Yes, I’m really proud of it, but I don’t want patents.” Cohen wasn’t sure whether basic scientific research funded by the government qualified for protection as private intellectual property, and wasn’t convinced that it should.
In response, Reimers emphasized the benefits of licensing arrangements to science and scientists. He explained that rights would be assigned to Stanford and UCSF, royalties would be paid only by commercial subscribers, and that, in his view, unlicensed technologies offered ‘free rides’ to users at the expense of universities and inventors. He also argued that without patent protection, many beneficial technologies would lie fallow in the literature. Cohen discussed the matter with Boyer. When Boyer indicated a willingness to proceed, Cohen gave his blessing.
On July 26, the participants in Berg’s MIT meeting published an editorial comment in Science that became known as the ‘Berg letter.’ It expressed “serious concern that artificial recombinant DNA molecules could prove biologically hazardous.” Berg and colleagues proposed a moratorium on certain kinds of recombinant DNA research until hazards and safety measures were assessed. Molecular biologists were asked to refrain from three types of experiments — those that entailed putting oncogenes, toxin genes, or drug resistance genes into E. coli. Planning began for a second meeting at Asilomar.
On November 4, 1974, in the face of a looming deadline, Reimers filed an application for a US patent on recombinant DNA technology. Under the ‘first to invent’ rules in force at the time, and thanks to a pre-Bayh-Dole Act institutional patent agreement with the NIH, Stanford had one year following disclosure of the technology to claim US property rights. The first Cohen-Boyer paper had appeared in print the previous November.
The application generated tensions within and between institutions of scientific research. At Stanford, Joshua Lederberg and Arthur Kornberg spoke out against the patent, arguing that the privatization of research would harm the scientific enterprise by compromising objectivity and encouraging secrecy.
In February, within three weeks of the second Asilomar meeting on biohazards in recombinant DNA research, a group of Stanford scientists led by Paul Berg and including David Hogness, Charles Yanofsky, and Ronald Davis met with Reimers to discuss a conflict of interest within the university: Stanford was actively pursuing the commercialization of recombinant DNA while faculty members were preparing to meet with academic colleagues to discuss risks associated with applications, and possibly efforts to restrict diffusion of the technology.
Berg also raised questions about the validity of the patent. In a meeting with Reimers, he asked, “Why is Cohen the only Stanford inventor? Many Stanford scientists contributed to DNA technology.” Berg claimed that work performed in his laboratory by Mertz and others constituted prior art that had already entered the public domain through scientific publications. “There was not,” says Reimers, “universal joy in the biochemistry establishment about what I was doing.”
When the patent examination got underway, Reimers asked the US Patent and Trademark Office (USPTO) to open the file of assembled evidence for public scrutiny. The process is ordinarily closed, but Reimers’s office was being overwhelmed by requests for information from pharmaceutical and chemical corporations. He was also by now immersed in internecine quarreling at the university, and aware that leading figures in the larger community of biological scientists opposed privatization and commercialization. He wanted to ensure transparency.
In “Tiger by the Tail,” a memoir and analysis of the episode published in 1987, Reimers explained the rationale for the unusual request: “Challenges to the patents in the courts seemed certain. Anyone who was aware of factors which would affect the patent’s validity was asked to make them known to the Patent Office. Any company seeking to challenge the validity of the patent after its issue would then have the burden of justifying why they had not raised those issues with the Patent Office before the patent issued.”
Bertram Rowland represented Stanford in the prosecution of the patent. After formulating the claims, he sent letters to co-authors on relevant scientific papers asking them to disclaim inventors’ rights. There were nine in all. “One agreed,” Rowland later wrote, “and the others either did not respond or sent unkind letters about the inventors and Stanford’s filing of the application.”
Shortly thereafter, the University of Michigan contested the patent, arguing that it was entitled to a share of royalties because faculty member Robert Helling, Boyer’s collaborator and a co-author on both papers, should have been designated a co-inventor of the technology. The challenge went nowhere — the legal definition of an inventor was well-defined, and the facts of the case were plainly demonstrable.
After an initial review, patent examiner Alvin E. Tanenholtz announced a blanket rejection of the application’s process claims. Following a review of evidence and arguments provided by Rowland in rebuttal, that decision was overturned, and each of the claims was allowed. Tanenholtz ultimately decided that Cohen and Boyer’s cloning system had combined existing technical elements in an original and novel way and had produced an original and novel outcome.
The ruling expressly excised hindsight bias from judgments regarding the obviousness of the work. There had been disagreements among ‘ordinary practitioners skilled in the art’ regarding whether and how gene cloning could be accomplished. The Cohen-Boyer experiments resolved outstanding questions.
While Tanenholtz accepted Cohen and Boyer’s process claims, he refused to grant protection to products made with the invention. Recombinant bacteria were living things. At that time, life was understood as a product of nature, and not human artifice. Rowland split the application, separating process and product claims so the former could be certified.
As Reimers learned how widely the invention could be applied, he came to realize that it was more than a method for manufacturing a specific set of end products — a class of drugs, for example. It was a powerful tool for engineering fundamental life processes. The possibilities were endless. Reimers saw that the invention would produce the greatest material good, not only for Stanford and UCSF, but for science and society, if widely disseminated and applied. He resolved to make recombinant DNA technology available to interested users through non-exclusive licenses offered at modest terms. The policy had enormous ramifications in the formation of the commercial biotechnology industry.
In January 1976, Boyer entered into a partnership with entrepreneur Robert Swanson, and a few months later established a private enterprise, Genentech, to put recombinant DNA technology to commercial ends. The company funded research in Boyer’s UCSF laboratory. When the arrangement became common knowledge at the university, a sizable faculty contingent expressed strong disapproval.
In 1980, Berg was awarded a share of the Nobel Prize in Chemistry for “fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA.” Cohen and Boyer were not recognized. The selection generated a great deal of speculation about the politics of disciplines, invisible colleges, and Nobel Prize review committees. Phil Sharp, who was present at the revolution and later became a Nobel Laureate himself for work performed in roughly the same period, notes that for some time after, “it was a lingering question as to why Boyer and Cohen never received a Nobel Prize for those experiments.”
It was a trying time for all involved. Eventually, the debates subsided and tensions eased. On March 17, 1980, the US Supreme Court ruled in Diamond v. Chakrabarty that living things were eligible for intellectual property protection. The USPTO agreed to review the application covering recombinant products.
The Cohen-Boyer process patent was granted on December 2, 1980. In August 1982, the NIH’s Recombinant DNA Advisory Committee issued revised and relaxed “Guidelines for Research Involving Recombinant DNA Molecules.” On August 28, 1984, the USPTO issued a patent on prokaryotic hosts of recombinant DNA. All was settled. The revolution was bureaucratically approved, and the world had been irrevocably changed.
— article by Mark Jones, PhD
To see more stories like this, visit us at biotechhistory.org
Learn more on our timeline of biotech history, with entries related to this article such as Recombinant DNA (1972), Recombinant DNA Technology (1973), and The “Berg Letter” (1974). Or see what Herb Boyer did next at Genentech (1976).
