The three components – cutting, pasting, and copying – came together in November 1972, in Honolulu. The occasion was a conference on plasmids. Herb Boyer, a newly tenured young professor at the University of California, San Francisco, was there, and, not surprisingly, so was Stanley Cohen, first among plasmid pioneers. Boyer, like Cohen, was an East Coast boy. A former high-school varsity lineman from western Pennsylvania, Boyer was perhaps fortunate that his football coach was also his science teacher. Like Cohen, he would be part of a new generation of scientists who were reared on the double helix. His enthusiasm for DNA even inspired him to name his Siamese cats Watson and Crick. No one, certainly not the coach, was surprised when after college he took up graduate work in bacterial genetics (see Plate 27).
Though Boyer and Cohen both now worked in the San Francisco Bay Area, they had not met before the Hawaii conference. Boyer was already an expert in restriction enzymes in an era when hardly anyone had even heard of them: it was he and his colleagues who had recently figured out the sequence of the cut site of the EcoRl enzyme. Boyer and Cohen soon realized that between them they had the skills to push molecular biology to a whole new level, the world of cut, paste, and copy. In a deli near Waikiki, they set about late one evening dreaming up the birth of recombinant DNA technology, jotting their ideas down on napkins. That visionary mapping of the future has been described as "from corned beef to cloning."
Within a few months, Boyer's lab in San Francisco and Cohen's forty miles to the south in Palo Alto were collaborating. Naturally Boyer's carried out the restriction enzyme work and Cohen's the plasmid procedures. Fortuitously a technician in Cohen's lab, Annie Chang, lived in San Francisco and was able to ferry the precious cargo of experiments in progress between the two sites. The first experiment intended to make a hybrid, "a recombinant," of two different plasmids, each of which was known to confer resistance to a particular antibiotic. On one plasmid there was a gene, a stretch of DNA, for resistance to tetracycline, and on the other a gene for resistance to kanamycin. (Initially, as we might expect, bacteria carrying the first type of plasmid were killed by kanamycin while those with the second were killed by tetracycline.) The goal was to make a single "super-plasmid" that would confer resistance to both.
First, the two types of unaltered plasmid were snipped with restriction enzymes (see Plate 28). Next the plasmids were mixed in the same test tube and ligase added to prompt the snipped ends to glue themselves together. For some molecules in the mix, the ligase would merely cause a snipped plasmid to make itself whole again – the two ends of the same plasmid would have been glued together. Sometimes, however, the ligase would cause a snipped plasmid to incorporate pieces of DNA from the other type of plasmid, thus yielding the desired hybrid. With this accomplished, the next step was to transplant all the plasmids into bacteria by using Cohen's plasmid-importing tricks. Colonies thus generated were then cultured on plates coated with both tetracycline and kanamycin. Plasmids that had simply re-formed would still confer resistance to only one of the antibiotics; bacteria carrying such plasmids would therefore not survive on the double-antibiotic medium. The only bacteria to survive were those with recombinant plasmids – those that had reassembled themselves from the two kinds of DNA present, the one coding for tetracycline resistance and the one coding for resistance to kanamycin.
The next challenge lay in creating a hybrid plasmid using DNA from a completely different sort of organism – a human being, for example. An early successful experiment involved putting a gene from the African clawed toad into an E. coli plasmid and transplanting that into bacteria. Every time cells in the bacterial colony divided, they duplicated the inserted segment of toad DNA. We had, in the rather confusing terminology of molecular biology, "cloned" the toad DNA. Mammal DNA, too, proved eminently clonable. This is not terribly surprising, in retrospect: a piece of DNA after all is finally still DNA, its chemical properties the same irrespective of its source. It was soon clear that Cohen and Boyer's protocols for cloning fragments of plasmid DNA would work just fine with DNA from any and every creature.
Phase 2 of the molecular biology revolution was thus under way. In phase 1 we aimed to describe how DNA works in the cell; now, with recombinant DNA,† we had the tools to intervene, to manipulate DNA. The stage was set for rapid progress, as we spied the chance to "play God." It was intoxicating: the extraordinary potential for delving deep into the mysteries of life and the opportunities for making real progress in the fight against diseases like cancer. But while Cohen and Boyer may indeed have opened our eyes to extraordinary scientific vistas, had they also opened a Pandora's box? Were there undiscovered perils in molecular cloning? Should we go on cheerfully inserting pieces of human DNA into E. coli, a species predominant in the microbial jungle in our guts? What if the altered forms should find their way into our bodies? In short, could we in good conscience simply turn a deaf ear to the cry of the alarmists, that we were creating bacterial Frankensteins?
*"Cloning" is the term applied to producing multiple identical pieces of a piece of DNA inserted into a bacterial cell The term is confusingly also applied to the cloning of whole animals, most notably Dolly the sheep. In the first type we are copying just a piece of DNA; in the other, we are copying an entire genome.
†The term "recombinant DNA" may present a little confusion in light of our encounter with "recombination" in the context of classical genetics. In Mendelian genetics, recombination involved the breaking and re-forming of chromosomes, with the result of a "mixing and matching" of chromosomal segments. In the molecular version, "mixing and matching" occurs on a much smaller scale, recombining two stretches of DNA into a single composite molecule.
In 1961 a monkey virus called SV40 ("SV" stands for "simian virus") was isolated from rhesus monkey kidneys being used for the preparation of polio vaccine. Although the virus was believed to have no effect on the monkeys in which it naturally occurs, experiments soon showed that it could cause cancer in rodents and, under certain laboratory conditions, even in human cells. Because the polio vaccination program had, since its inception in 1955, infected millions of American children with the virus, this discovery was alarming indeed. Had the polio prevention program inadvertently condemned a generation to cancer? The answer, fortunately, seems to be "no"; no epidemic of cancer has resulted, and SV40 seems to be no more pernicious in living humans than it is in monkeys. Nevertheless, even as SV40 was becoming a fixture in molecular biology laboratories, there remained doubts about its safety. I was particularly concerned since I was by this time head of the Cold Spring Harbor Laboratory, where growing ranks of young scientists were working with SV40 to probe the genetic basis of cancer.
Meanwhile, at Stanford University Medical School, Paul Berg was more excited by the promise than by the dangers of SV40; he foresaw the possibility of using the virus to introduce pieces of DNA – foreign genes – into mammalian cells. The virus would work as a molecular delivery system in mammals, just as plasmids had been put to work in bacteria by Stanley Cohen. But whereas Cohen used bacteria essentially as copy machines, which could amplify up a particular piece of DNA, Berg saw in SV40 a means to introduce corrective genes into the victims of genetic disease. Berg was ahead of his time. He aspired to carry out what today is called gene therapy: introducing new genetic material into a living person to compensate for inherited genetic flaws.
Berg had come to Stanford as a junior professor in 1959 as part of the package deal that also brought the more eminent Arthur Kornberg there from Washington University in St. Louis. In fact, Berg's connections to Kornberg can be traced all the way back to their common birthplace of Brooklyn, New York, where each in his time was to pass through the same high-school science club run by a Miss Sophie Wolfe. Berg recalled: "She made science fun, she made us share ideas." It was an understatement really: Miss Wolfe's science club at Abraham Lincoln High School would produce three Nobel laureates – Romberg (1959), Berg (1980), and the crystallographer Jerome Karle (1985) – all of
whom have paid tribute to her influence.
While Cohen and Boyer, and by now others, were ironing out the details of how to cut and paste DNA molecules, Berg planned a truly bold experiment: he would see whether SV40, implanted with a piece of DNA not its own, could be made to transport that foreign gene into an animal cell. For convenience he would use as the source of his non-SV40 DNA a readily available bacterial virus, a bacteriophage. The aim was to see whether a composite molecule consisting of SV40 DNA and the bacteriophage DNA could successfully invade an animal cell. If it could, as Berg hoped, then the possibility existed that he could ultimately use this system to insert useful genes into human cells.
At Cold Spring Harbor Laboratory in the summer of 1971, a graduate student of Berg's gave a presentation explaining the planned experiment. One scientist in the audience was alarmed enough to phone Berg straightaway. What if, he asked, things happened to work in reverse? In other words, what if the SV40 virus, rather than taking up the viral DNA and then inserting it into the animal cell, was itself manipulated by the bacteriophage DNA, which might cause the SV40 DNA to be inserted into, say, an E. coli bacterial cell? It was not an unrealistic scenario: after all, that is precisely what many bacteriophages are programmed to do – to insert their DNA into bacterial cells. Since E. coli is both ubiquitous and intimately associated with humans, as the major component of our gut flora, Berg's well-meaning experiment might result in dangerous colonies of E. coli carrying SV40 monkey virus, a potential cancer agent. Berg heeded his colleague's misgivings, though he did not share them: he decided to postpone the experiments until more could be learned about SV40's potential to cause human cancer (see Plate 29).
Biohazard anxieties followed hard on the heels of the news of Boyer and Cohen's success with their recombinant DNA procedures. At a scientific conference on nucleic acids in New Hampshire in the summer of 1973, a majority voted to petition the National Academy of Sciences to investigate without delay the dangers of the new technology. A year later a committee appointed by the National Academy and chaired by Paul Berg published its conclusions in a letter to the journal Science. I myself signed the letter, as did many of the others – including Cohen and Boyer – who were most active in the relevant research. In what has since come to be known as the "Moratorium Letter" we called upon "scientists throughout the world" to suspend voluntarily all recombinant studies "until the potential hazards of such recombinant DNA molecules have been better evaluated or until adequate methods are developed for preventing their spread." An important element of this statement was the admission that "our concern is based on judgements of potential rather than demonstrated risk since there are few experimental data on the hazards of such DNA molecules."
All too soon, however, I found myself feeling deeply frustrated and regretful of my involvement in the Moratorium Letter. Molecular cloning had the obvious potential to do a fantastic amount of good in the world, but now, having worked so hard and arrived at the brink of a biological revolution, here we were conspiring to draw back. It was a confusing moment. As Michael Rogers wrote in his 1975 report on the subject for Rolling Stone, "The molecular biologists had clearly reached the edge of an experimental precipice that may ultimately prove equal to that faced by nuclear physicists in the years prior to the atom bomb." Were we being prudent or chickenhearted? I couldn't quite tell yet, but I was beginning to feel it was the latter.
The "Pandora's Box Congress": that's how Rogers described the February 1975 meeting of 140 scientists from around the world at the Asilomar conference center in Pacific Grove, California. The agenda was to determine once and for all whether recombinant DNA really held more peril than promise. Should the moratorium be permanent? Should we press ahead regardless of potential risk, or wait for the development of certain safeguards? As chair of the organizing committee, Paul Berg was also nominal head of the conference, and so had the almost impossible task of drafting a consensus statement by the end of the meeting.
The press was there, scratching its collective head as scientists bandied about the latest jargon. The lawyers were there, too, just to remind us that there were also legal issues to be addressed: for example, would I, as head of a lab doing recombinant research, be liable if a technician of mine developed cancer? As to the scientists, they were by nature and training averse to hazarding predictions in the absence of knowledge; they rightly suspected that it would be impossible to reach a unanimous decision. Perhaps Berg was equally doubtful; in any case, he opted for freedom of expression over firm leadership from the chair. The resulting debate was therefore something of a free-for-all, with the proceedings not infrequently derailed by some speaker intent only on rambling irrelevantly and at length about the important work going on in his or her lab. Opinions ranged wildly, from the timid – "prolong the moratorium" – to the gung ho – "the moratorium be damned, let's get on with the science." I was definitely on the latter end of the spectrum. I now felt that it was more irresponsible to defer research on the basis of unknown and unquantified dangers. There were desperately sick people out there, people with cancer or cystic fibrosis – what gave us the right to deny them perhaps their only hope?
Sydney Brenner, then based in the United Kingdom, at Cambridge, offered one of the very few pieces of relevant data. He had collected colonies of the E. coli strain known as K-12, the favorite bacterial workhorse for this kind of molecular cloning research. Particular rare strains of E. coli occasionally cause outbreaks of food poisoning, but in fact the vast majority of E. coli strains are harmless, and Brenner assumed that K-12 was no exception. What interested him was not his own health but K-12's: could it survive outside the laboratory? He stirred the microbes into a glass of milk (they were rather unpalatable served up straight), and went on to quaff the vile mixture. He monitored what came out the other end to see whether any K-12 cells had managed to colonize his intestine. His finding was negative, suggesting that K-12, despite thriving in a petri dish, was not viable in the "natural" world. Still, others questioned the inference: even if the K-12 bacteria were themselves unable to survive, this was no proof they could not exchange plasmids – or other genetic information – with strains that could live perfectly well in our guts. Thus "genetically engineered" genes could still enter the population of intestine-dwelling bacteria. Brenner then championed the idea that we should develop a K-12 strain that was without question incapable of living outside the laboratory. We could do this by a genetic alteration that would ensure the strain could grow only when supplied with specialized nutrients. And of course we would specify a set of nutrients that would never be available in the natural world; the full complement of nutrients would occur together only in the lab. A K-12 thus modified would be a "safe" bacterium, viable in our controlled research setting, but doomed in the real world.
With Brenner's urging, this middle-ground proposal carried the day. There was plenty of grumbling from both extremes, of course, but the conference ended with coherent recommendations allowing research to continue on disabled, non-disease-causing bacteria and mandating expensive containment facilities for work involving the DNA of mammals. These recommendations would form the basis for a set of guidelines issued a year later by the National Institutes of Health.
I departed feeling despondent, isolated from most of my peers. Stanley Cohen and Herb Boyer found the occasion disheartening as well; they believed, as I did, that many of our colleagues had compromised their better judgment as scientists just to be seen by the assembled press as "good guys" (and not as potential Dr. Frankensteins). In fact, the vast majority had never worked with disease-causing organisms and little understood the implications of the research restrictions they wanted to impose on those of us who did. I was irked by the arbitrariness of much of what had been agreed: DNA from cold-blooded vertebrates was, for instance, deemed acceptable, while mammalian DNA was ruled off-limits for most scientists. Apparently it was safe to work with DNA from a toad but not with DNA from a mouse. Dumbstruck by such nonsense, I offered u
p a bit of my own: didn't everyone know that toads cause warts? But my facetious objections were in vain.
The guidelines led many participants in the Asilomar conference to expect clear sailing for research based on cloning in "safe bacteria." But anyone who set off under such an impression very soon hit choppy seas. According to the logic peddled by the popular press, if scientists themselves saw cause for concern, then the public at large should really be alarmed. These were, after all, still the days, though waning, of the American counterculture. Both the Vietnam War and Richard Nixon's political career had only recently petered out; a suspicious public, ill-equipped to understand complexities that science itself was only beginning to fathom, was only too eager to swallow theories of evil conspiracies perpetrated by the Establishment. For our part, we scientists were quite surprised to see ourselves counted among this elite, to which we had never before imagined we belonged. Even Herb Boyer, the veritable model of a hippie scientist, would find himself named in the special Halloween issue of the Berkeley Barb, the Bay Area's underground paper, as one of the region's "ten biggest bogeymen," a distinction otherwise reserved for corrupt pols and union-busting capitalists.
Dna: The Secret of Life Page 11
