Paul Berg, Herbert W. Boyer, and Stanley N. Cohen

Paul Berg opening a jar under a protective hood. Courtesy Stanford University Archives.

Paul Berg opening a jar under a protective hood. Courtesy Stanford University Archives.

The invention of recombinant DNA technology—the way in which genetic material from one organism is artificially introduced into the genome of another organism and then replicated and expressed by that other organism—was largely the work of Paul Berg (b. 1926), Herbert W. Boyer (b. 1936), and Stanley N. Cohen (b. 1935), although many other scientists made important contributions to the new technology as well.

Berg grew up in Brooklyn, New York, in the 1930s. His interest in science was stimulated by his reading of Paul De Kruif’s Microbe Hunters (1926) and Sinclair Lewis’s Arrowsmith (1925). After graduating from high school at the age of 16, he had some difficulty deciding what and where he should study. Perusal of a catalog from Pennsylvania State College (now University) alerted him to the existence of the field of biochemistry, and he was soon on his way to Penn State. In the middle of his college years his enlistment in the U.S. Navy was activated, but World War II ended before he saw combat. Berg returned to Penn State to complete his degree, then went on to Western Reserve University (now Case Western Reserve) to get a Ph.D. in biochemistry. He pursued postdoctoral research on enzymes and in 1955 was appointed to the faculty at Washington University, where he became a leader in deciphering the biosynthesis of proteins on the basis of codes carried on deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules (see Maxine Singer).

>Figure 1. The process of making recombinant DNA, as pioneered by Paul Berg.

Figure 1. The process of making recombinant DNA, as pioneered by Paul Berg.

In 1959 Berg joined the faculty of Stanford University. There he became interested in the genetics of microbes and took a leave to study at Renato Dulbecco’s laboratory at the Salk Institute, where he learned the techniques of animal-cell culture. Dulbecco had already shown that certain viruses induce a cancerous state in an infected cell by taking over the expression of the genetic information of that cell for their own reproduction. Like other scientists at the time, Berg began to wonder whether it would be possible to insert foreign genes into a virus, thereby causing it to become the vector by which genes could be carried into new cells.

Berg’s 1971 landmark gene-splicing experiment (Figure 1) involved splicing a bit of the DNA of the bacterial virus known as lambda into the DNA of simian virus SV40, whose natural host is the monkey. The DNA of both these viruses occurs in closed loops. In the first step of Berg’s experiment the loops were each cut in one place by an enzyme, EcoRI. Next, to make the ends of these now-linear molecules stick together again, they were modified by two other enzymes using a procedure developed by Stanford colleagues. Then the two types of DNA were mixed together where they rejoined into loops in such a way that the new loops combined DNA from each source. Berg’s gene-splicing experiment resulted in the first man-made recombinant DNA (rDNA), as such molecules came to be called. The award ceremony for Berg’s 1980 Nobel Prize in chemistry, shared with Walter Gilbert and Frederick Sanger, highlighted this work.

Herbert Boyer at the Chemical Heritage Foundation in 2005. Photograph by Douglas Lockard. CHF Collections.

Herbert Boyer at the Chemical Heritage Foundation in 2005. Photograph by Douglas Lockard. CHF Collections.

Stanley N. Cohen. Courtesy Stanford University Archives.

Stanley N. Cohen. Courtesy Stanford University Archives.

Berg did not immediately take the step of introducing the rDNA into another organism because of the public controversy over the potential dangers of such experimentation. The fear was that rDNA carrying a dreaded gene—for example, for the creation of cancerous tumors—might escape the laboratory in some common bacteria and be spread everywhere. As chair of the National Academy of Science’s Committee on Recombinant DNA Molecules, Berg played an active role in the debate among scientists and with the public about potential limitations on such research. In the 1970s the National Institutes of Health issued guidelines for the safe conduct of rDNA research. Over time these guidelines have been eased, as more experience has shown the hazards to be far less than imagined.

The next landmark in the development of modern biotechnology was the insertion of rDNA into bacteria in such a way that the foreign DNA would replicate naturally (Figure 2). This step was taken in 1972 by Herbert Boyer at the University of California at San Francisco (UCSF), in collaboration with Stanley Cohen of Stanford University.

Born and raised in western Pennsylvania, Boyer attended St. Vincent’s College in Latrobe, where he enrolled in premedical studies. He was soon captivated by research, however, and chose to major in chemistry and biology. He completed a Ph.D. in biochemistry at the University of Pittsburgh and then went on to a postdoctoral fellowship at Yale University. In 1966 he accepted an appointment at UCSF, which was becoming a center of excellence in the several disciplines that contributed to the emerging field of biotechnology (see William J. Rutter).

Figure 2. The insertion of recombinant DNA so that the foreign DNA will replicate naturally, as pioneered by Herbert Boyer and Stanley Cohen.

Figure 2. The insertion of recombinant DNA so that the foreign DNA will replicate naturally, as pioneered by Herbert Boyer and Stanley Cohen.

In 1972 researchers, including Boyer, realized that the enzyme EcoRI, which had actually been discovered in Boyer’s UCSF lab, cut DNA in such a way that the ends were not blunt but staggered, so that no molecular additions were needed to make one severed piece latch on to another piece possessing complementary cuts. Boyer and a colleague, Robert Helling, began their effort to create rDNA to insert in the bacteria Escherichia coli (E. coli) by trying to use EcoRI to open up the DNA of the bacterial virus lambda. They became frustrated, however, when the enzyme cut the DNA in five places instead of one, as desired.

November 1972 found both Boyer and Cohen in Hawaii giving papers at a U.S.-Japan joint meeting on plasmids. A plasmid is DNA, found especially in bacteria, that is physically separate from, and can replicate independently of, the bacterium’s chromosomal DNA. While Boyer was describing his data showing the nature of the DNA ends generated by EcoRI cleavage, Cohen was reporting on a procedure recently discovered in his laboratory that enabled bacteria to take up plasmid DNA and produce offspring that contained self-replicating plasmids identical to the original implant—clones. Over sandwiches late one night at the conference, the two men laid plans for a collaborative project to discover what genes are present on plasmids and how they are arranged.

Stanley N. Cohen. Courtesy Stanford University Archives.

Stanley N. Cohen. Courtesy Stanford University Archives.

A native of Perth Amboy, New Jersey, Cohen received his undergraduate education at Rutgers University and then proceeded to the University of Pennsylvania for an M.D. After completing his medical education, he began a full-time career in medical research and teaching at the Albert Einstein College of Medicine in New York. There he worked on the complex mechanisms that control gene expression in the bacterial virus lambda. In 1968 he accepted an appointment at the Stanford University School of Medicine.

The collaboration between Boyer and Cohen was very close. Plasmids isolated at Stanford were transported to Boyer’s lab in San Francisco for cutting by EcoRI and for analysis of the DNA fragments. These were transported back to Stanford, where they were joined and introduced into E. coli, where they multiplied. Then the brand-new recombinant plasmids were isolated and analyzed in each laboratory.

The first success of the Boyer-Cohen collaboration occurred in spring 1973 and involved one of Cohen’s plasmids, pSC101. Plasmids were already known to transfer drug resistance among bacteria, and this one could make E. coli resistant to the antibiotic tetracycline. The plasmid pSC101 was cleaved by EcoRI at only one site, leaving intact the plasmid’s ability to replicate. When the linearized pSC101 DNA was mixed with other DNA that had been cleaved by the same enzyme, the complementary ends of fragments from both sources of DNA joined together into new loops. Treatment with another enzyme closed the still-visible nicks in the DNA loops, which were then introduced into calcium chloride–treated bacteria. The bacteria were spread on a culture containing tetracycline, and only the bacteria with the rDNA plasmids survived.

Boyer and Cohen soon moved on to more complicated cloning experiments. They joined tetracycline-resistant plasmids with kanamycin-resistant plasmids—kanamycin being another antibiotic—and inserted them in E. coli. Next they showed that genetic materials could indeed be transferred between species, thereby disproving a long-held myth. They snipped a piece of Staphylococcus plasmid (Staphylococcus is the bacteria responsible for Staph infections), and spliced it with one of the many E. coli plasmids and inserted the whole in E. coli. The DNA from Staphylococcus, a different species of bacteria, was successfully propagated in E. coli. An even-greater triumph of interspecies cloning was the insertion into E. coli of genes taken from the South African clawed frog.

Commercial ventures quickly started up with the objective of capitalizing on Boyer and Cohen’s new recombinant DNA technology, despite ongoing controversy over the technology and public fear of cloning. At the forefront was Genentech, founded in 1976 by Boyer and a young venture capitalist, Robert Swanson.

Boyer and Cohen, as well as other scientists involved in cloning experimentation, soon recognized the feasibility of using bacteria into which human genetic information was incorporated to duplicate the body’s natural means of fighting disease and to remedy birth disorders. In fall 1977, even before Genentech had its own facilities, Boyer at UCSF and Keiichi Itakura at the City of Hope Medical Center in Duarte, California, succeeded in expressing a mammalian protein in bacteria—somatostatin. This hormone, produced in the human brain, plays a major role in regulating the growth hormone. Recombinant somatostatin was shown to be virtually identical to the naturally occurring substance.

In 1978 Boyer and Itakura also constructed a plasmid that coded for human insulin. By then they had many rivals, some of them small start-ups backed by large pharmaceutical companies. In the case of recombinant insulin, Eli Lilly and Company signed a joint-venture agreement with Genentech to develop the production process for Humulin. In 1982 Humulin was approved by the FDA, and it became the first biotechnology product to appear on the market.

Whereas Boyer became deeply involved in this commercial work, Cohen remained an academic researcher, focusing on basic questions in genetics and biology. Over the years, however, Cohen has consulted for several biotechnology companies, as did Berg, who later helped found a company called DNAX.

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