The cutting enzymes came from a more unusual source. Virtually all cells have ligases and polymerases to repair broken DNA, but there is little reason for most cells to have a DNA-cutting enzyme on the loose. But bacteria and viruses—organisms that live on the roughest edges of life, where resources are drastically limited, growth is fierce, and competition for survival is intense—possess such knifelike enzymes to defend themselves against each other. They use DNA-cutting enzymes, like switchblades, to slice open the DNA of invaders, thereby rendering their hosts immune to attack. These proteins are called “restriction” enzymes because they restrict infections by certain viruses. Like molecular scissors, these enzymes recognize unique sequences in DNA and cut the double helix at very specific sites. The specificity is key: in the molecular world of DNA, a targeted gash at the jugular can be lethal. One microbe can paralyze an invading microbe by cutting its chain of information.
These enzymatic tools, borrowed from the microbial world, would form the basis of Berg’s experiment. The crucial components to engineer genes, Berg knew, were frozen away in about five separate refrigerators in five laboratories. He just needed to walk to the labs, gather the enzymes, and string the reactions in a chain. Cut with one enzyme, paste with another—and any two fragments of DNA could be stitched together, allowing scientists to manipulate genes with extraordinary dexterity and skill.
Berg understood the implications of the technology that was being created. Genes could be combined to create new combinations, or combinations of combinations; they could be altered, mutated, and shuttled between organisms. A frog gene could be inserted into a viral genome and thus introduced into a human cell. A human gene could be shuttled into bacterial cells. If the technology was pushed to its extreme limits, genes would become infinitely malleable: you could create new mutations or erase them; you could even envision modifying heredity—washing its marks, cleaning it, changing it at will. To produce such genetic chimeras, Berg recalled, “none of the individual procedures, manipulations, and reagents used to construct this recombinant DNA was novel; the novelty lay in the specific way they were used in combination.” The truly radical advance was the cutting and pasting of ideas—the reassortment and annealing of insights and techniques that already existed in the realm of genetics for nearly a decade.
In the winter of 1970, Berg and David Jackson, a postdoctoral researcher in Berg’s lab, began their first attempts to cut and join two pieces of DNA. The experiments were tedious—“a biochemist’s nightmare,” as Berg described them. The DNA had to be purified, mixed with the enzymes, then repurified on ice-cold columns, and the process repeated, until each of the individual reactions could be perfected. The problem was that the cutting enzymes had not been optimized, and the yield was minuscule. Although preoccupied with his own construction of gene hybrids, Lobban continued to provide crucial technical insights to Jackson. He had found a method to add fragments to the ends of DNA to make two clasplike pieces that stuck together like latch and key, thereby vastly increasing the efficiency with which gene hybrids could be formed.
Despite the forbidding technical hurdles, Berg and Jackson managed to join the entire genome of SV40 to a piece of DNA from a bacterial virus called Lambda bacteriophage (or phage λ) and three genes from the bacterium E. coli.
This was no mean achievement. Although λ and SV40 are both “viruses,” they are as different from each other, say, as a horse and a seahorse (SV40 infects primate cells, while phage λ only infects bacteria). And E. coli was an altogether different beast—a bacterium from the human intestine. The result was a strange chimera: genes from far branches of the evolutionary tree stitched together to form a single contiguous piece of DNA.
Berg called the hybrids “recombinant DNA”. It was a cannily chosen phrase, harkening back to the natural phenomenon of “recombination,” the genesis of hybrid genes during sexual reproduction. In nature, genetic information is frequently mixed and matched between chromosomes to generate diversity: DNA from the paternal chromosome swaps places with DNA from the maternal chromosome to generate “father:mother” gene hybrids—“crossing over,” as Morgan had called the phenomenon. Berg’s genetic hybrids, produced with the very tools that allowed genes to be cut, pasted, and repaired in their natural state in organisms, extended this principle beyond reproduction. Berg was also synthesizing gene hybrids, albeit with genetic material from different organisms, mixed and matched in test tubes. Recombination without reproduction: he was crossing over to a new cosmos of biology.
Figure adapted from Paul Berg’s paper on “Recombinant” DNA. By combining genes from any organisms, scientists could engineer genes at will, foreshadowing human gene therapy and human genome engineering.
That winter, a graduate student named Janet Mertz decided to join Berg’s lab. Tenacious, unabashedly vocal about her opinions—“smart as all hell,” as Berg described her—Mertz was an anomaly in the world of biochemists: the second woman to join Stanford’s biochemistry department in nearly a decade. Like Lobban, Mertz had also come to Stanford from MIT, where she had majored in both engineering and biology. Mertz was intrigued by Jackson’s experiments and was keen on the idea of synthesizing chimeras between genes of different organisms.
But what if she inverted Jackson’s experimental goal? Jackson had inserted genetic material from a bacterium into the SV40 genome. What if she made genetic hybrids with SV40 genes inserted into the E. coli genome? Rather than viruses carrying bacterial genes, what might happen if Mertz created bacteria carrying viral genes?
The inversion of logic—or rather, the inversion of organisms—carried a crucial technical advantage. Like many bacteria, E. coli carry minuscule extra chromosomes, called mini-chromosomes or plasmids. As with the SV40 genome, plasmids also exist as circular necklaces of DNA, and they live and replicate within the bacteria. As bacterial cells divide and grow, the plasmids are also replicated. If Mertz could insert SV40 genes into an E. coli plasmid, she realized, she could use the bacteria as a “factory” for the new gene hybrids. As the bacteria grew and divided, the plasmid—and the foreign gene inside it—would be amplified manyfold. Copy upon copy of the modified chromosome, and its payload of foreign genes, would be created by the bacteria. There would ultimately be millions of exact replicas of a piece of DNA—“clones.”
In June 1972, Mertz traveled from Stanford to Cold Spring Harbor in New York to attend a course on animal cells and viruses. As part of the course, students were expected to describe the research projects that they wished to pursue in the future. During her presentation, Mertz spoke about her plans to make genetic chimeras of SV40 and E. coli genes, and potentially propagate these hybrids in bacterial cells.
Graduate talks in summer courses typically don’t generate much excitement. By the time Mertz was done with her slides, though, it was clear that this wasn’t a typical graduate talk. There was silence at the end of Mertz’s presentation—and then the students and instructors broke upon her with a tidal wave of questions: Had she contemplated the risks of generating such hybrids? What if the genetic hybrids that Berg and Mertz were about to generate were let loose on human populations? Had they considered the ethical aspects of making novel genetic elements?
Immediately after the session, Robert Pollack, a virologist and an instructor at the course, called Berg urgently. Pollack argued that the dangers implicit in “bridging evolutionary barriers that had existed since the last common ancestors between bacterium and people” were far too great to continue the experiment casually.
The issue was particularly thorny because SV40 was known to cause tumors in hamsters, and E. coli was known to live in the human intestine (current evidence suggests that SV40 is not likely to cause cancer in humans, but the risks were still unknown in the 1970s). What if Berg and Mertz ended up concocting the perfect storm of a genetic catastrophe—a human intestinal bacterium carrying a human cancer-causing gene? “You can stop splitting the atom; you can stop visiting the moon; you can stop using aer
osol. . . . But you cannot recall a new form of life,” Erwin Chargaff, the biochemist, wrote. “[The new genetic hybrids] will survive you and your children and your children’s children. . . . The hybridization of Prometheus with Herostratus is bound to give evil results.”
Berg spent weeks deliberating over the concerns raised by Pollack and Chargaff. “My first reaction was: this was absurd. I didn’t really see any risk to it.” The experiments were being carried out in a contained facility, with sterilized equipment; SV40 had never been implicated directly in human cancers. Indeed, many virologists had become infected with SV40, and no one had acquired any cancers. Frustrated with the constant public hysteria around the issue, Dulbecco had even offered to drink SV40 to prove that there was no link to human cancers.
But with his feet slung on the edge of a potential precipice, Berg could not afford to be cavalier. He wrote to several cancer biologists and microbiologists, asking them for independent opinions of the risk. Dulbecco was adamant about SV40, but could any scientist realistically estimate an unknown risk? In the end, Berg concluded that the biohazard was extremely minimal—but not zero. “In truth, I knew the risk was little,” Berg said. “But I could not convince myself that there would be no risk. . . . I must have realized that I’d been wrong many, many times in predicting the outcomes of an experiment, and if I was wrong about the outcome of the risk, then the consequences were not something that I would want to live with.” Until he had determined the precise nature of the risk, and made a plan for containment, Berg placed a self-imposed moratorium. For now, the DNA hybrids containing pieces of the SV40 genome would remain in a test tube. They would not be introduced into living organisms.
Mertz, meanwhile, had made another crucial discovery. The initial cutting and pasting of DNA, as envisaged by Berg and Jackson, required six tedious enzymatic steps. Mertz found a useful shortcut. Using a DNA-cutting enzyme—called EcoR1—obtained from Herb Boyer, a microbiologist in San Francisco, Mertz found that the pieces could be cut and pasted together in just two steps, rather than six.II “Janet really made the process vastly more efficient,” Berg recalled. “Now, in just a few chemical reactions, we could generate new pieces of DNA. . . . She cut them, mixed them, added an enzyme that could join ends to ends, and then showed that she had gotten a product that shared the properties of both the starting materials.”
In November 1972, while Berg was weighing the risks of virus-bacteria hybrids, Herb Boyer, the San Francisco scientist who had supplied the DNA-cutting enzymes to Mertz, traveled to Hawaii for a meeting on microbiology. Born in a mining town in Pennsylvania in 1936, Boyer had discovered biology as a high school student and had grown up idealizing Watson and Crick (he had named his two Siamese cats after them). He had applied to medical school in the early sixties, but was rejected, unable to live down a D in metaphysics; instead, he had switched to studying microbiology as a graduate student.
Boyer had arrived in San Francisco in the summer of ’66—with an afro, the requisite leather vest, and cutoff jeans—as an assistant professor at University of California, San Francisco (UCSF). Much of his work concerned the isolation of novel DNA-cutting enzymes, such as the one that he had sent to Berg’s lab. Boyer had heard from Mertz about her DNA-cutting reaction, and the consequent simplification of the process of generating DNA hybrids.
The conference in Hawaii was about bacterial genetics. Much of the excitement at the meeting involved the newly discovered plasmids in E. coli—the circular mini-chromosomes that replicated within bacteria, and could be transmitted between bacterial strains. After a long morning of presentations, Boyer fled to the beach for a respite, and spent the afternoon nursing a glass of rum and coconut juice.
Late that evening, Boyer ran into Stanley Cohen, a professor at Stanford. Boyer knew Cohen from his scientific papers, but they had never met in person. With a neatly trimmed, graying beard, owlish spectacles, and a cautious, deliberate manner of speaking, Cohen had the “physical persona of a Talmudic scholar,” one scientist recalled—and a Talmudic knowledge of microbial genetics. Cohen worked on plasmids. He was also an expert on Frederick Griffith’s “transformation” reaction—the technique needed to deliver DNA into bacterial cells.
Dinner had ended, but Cohen and Boyer were still hungry. With Stan Falkow, a fellow microbiologist, they strolled out of the hotel toward a quiet, dark street in a commercial strip near Waikiki beach. A New York–style deli, with bright flashing signs and neon-lit fixtures, loomed providentially out of the shadows of the volcanoes, and they found an open booth inside it. The waiter couldn’t tell a kishke from a knish, but the menu offered corned beef and chopped liver. Over pastrami sandwiches, Boyer, Cohen, and Falkow talked about plasmids, gene chimeras, and bacterial genetics.
Both Boyer and Cohen knew about Berg’s attempts to create gene-hybrids in the lab. Cohen also knew that Mertz, Berg’s graduate student, was making the rounds among the microbiologists at Stanford, seeking to learn techniques to transfer her novel gene hybrids into E. coli.
The discussion moved casually to Cohen’s work. Cohen had isolated several plasmids from E. coli, including one that could be reliably purified out of the bacteria, and easily transmitted from one E. coli strain into another. Some of these plasmids carried genes to confer resistance to antibiotics—to tetracycline or penicillin, say.
But what if Cohen cut out an antibiotic-resistance gene from one plasmid and shuttled it to another plasmid? Wouldn’t a bacterium previously killed by the antibiotic now survive, thrive, and grow selectively, while the bacteria carrying the non-hybrid plasmids would die?
The idea flashed out of shadows, like a neon sign on a darkening island. In Berg’s and Jackson’s initial experiments, there had been no simple method to identify the bacteria or viruses that had acquired the “foreign” gene (the hybrid plasmid had to be purified out of the biochemical gumbo using its size alone: A + B was larger than A or B). Cohen’s plasmids, carrying antibiotic-resistance genes, in contrast, provided a powerful means to identify genetic recombinants. Evolution would be conscripted to help their experiment. Natural selection, deployed in a petri dish, would naturally select their hybrid plasmids. The transference of antibiotic resistance from one bacterium to another bacterium would confirm that the gene hybrid, or recombinant DNA, had been created.
But what of Berg and Jackson’s technical hurdles? If the genetic chimeras were produced at a one-in-a-million frequency, then no selection method, however deft or powerful, would work: there would be no hybrids to select. On a whim, Boyer began to describe the DNA-cutting enzymes and Mertz’s improved process to generate gene hybrids with greater efficiency. There was silence, as Cohen and Boyer tossed the idea around in their minds. The convergence was inevitable. Boyer had purified enzymes to create gene hybrids with vastly improved efficiency; Cohen had isolated plasmids that could be selected and propagated easily in bacteria. “The thought,” Falkow recalls, was “too obvious to slip by unnoticed.”
Cohen spoke in a slow, clear voice: “That means—”
Boyer cut him off mid-thought: “That’s right . . . it should be possible. . . .”
“Sometimes in science, as in the rest of life,” Falkow later wrote, “it is not necessary to finish the sentence or thought.” The experiment was straightforward enough—so magnificently simple that it could be performed over the course of a single afternoon with standard reagents: “mix EcoR1-cut plasmid DNA molecules and rejoin them and there should be a proportion of recombinant plasmid molecules. Use antibiotic resistance to select the bacteria that had acquired the foreign gene, and you would select the hybrid DNA. Grow one such bacterial cell into its million descendants, and you would amplify the hybrid DNA a millionfold. You would clone recombinant DNA.”
The experiment was not just innovative and efficient; it was also potentially safer. Unlike Berg and Mertz’s experiment—involving virus-bacteria hybrids—Cohen and Boyer’s chimeras were composed entirely of bacterial genes, which they considered far
less hazardous. They could find no reason to halt the creation of these plasmids. Bacteria, after all, were capable of trading genetic material like gossip, with scarcely an afterthought; free trade in genes was a hallmark of the microbial world.
Through that winter, and into the early spring of 1973, Boyer and Cohen worked furiously to make their genetic hybrids. Plasmids and enzymes shuttled between UCSF and Stanford, up and down Highway 101, on a Volkswagen Beetle driven by a research assistant from Boyer’s lab. A few thousand feet from Cohen’s lab, Berg and Mertz were also working on their experiments. They knew distantly about Cohen’s forays into recombinant DNA, but their own efforts were still focused on optimizing the reaction in a test tube. By the end of the summer, Boyer and Cohen had successfully created their gene hybrids—two pieces of genetic material from two bacteria stitched together to form a single chimera. Boyer later recalled the moment of discovery with immense clarity: “I looked at the first gels and I remember tears coming into my eyes, it was so nice.” Hereditary identities borrowed from two organisms had been shuffled around to form a new one; it was as close to metaphysics as one could get.
In February 1973, Boyer and Cohen were ready to propagate the first artificially produced genetic chimera in living cells. They cut two bacterial plasmids open with restriction enzymes and swapped the genetic material from one plasmid into another. The plasmid carrying the hybrid DNA was locked shut with ligase, and the resultant chimera introduced into bacterial cells using a modified version of the transformation reaction. The bacteria containing the gene hybrids were grown on petri dishes to form tiny translucent colonies, glistening like pearls on agar.
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