My greatest fear was that this blooming public paranoia about molecular biology would result in draconian legislation. Having experimental dos and don'ts laid down for us in some cumbersome legalese could only be bad for science. Plans for experiments would have to be submitted to politically minded review panels, and the whole hopeless bureaucracy that comes with this kind of territory would take hold like the moths in Grandmother's closet. Meanwhile, our best attempts to assess the real risk potential of our work continued to be dogged by a complete lack of data and by the logical difficulty of proving a negative. No recombinant DNA catastrophe had ever occurred, but the press continued to outdo itself imagining "worst case scenarios." In his account of a meeting in Washington, D.C., in 1977, the biochemist Leon Heppel aptly summed up the absurdities scientists perceived in the controversy.
I felt the way 1 would feel if I had been selected for an ad hoc committee convened by the Spanish Government to try to evaluate the risks assumed by Christopher Columbus and his sailors, a committee that was supposed to set up guidelines for what to do in case the earth was flat, how far the crew might safely venture to the earth's edge, etc.
Even withering irony, however, could little hinder those hell-bent on countering what they saw as science's Promethean hubris. One such crusader was Alfred Vellucci, the mayor of Cambridge, Massachusetts. Vellucci had earned his political chops championing the common man at the expense of his town's elite institutions of learning, namely, MIT and Harvard. The recombinant DNA tempest provided him with a political bonanza. A contemporary account captures nicely what was going on.
In his cranberry doubleknit jacket and black pants, with his yellow-striped blue shirt struggling to contain a beer belly, right down to his crooked teeth and overstuffed pockets, Al Vellucci is the incarnation of middle-American frustration at these scientists, these technocrats, these smartass Harvard eggheads who think they've got the world by a string and wind up dropping it in a puddle of mud. And who winds up in the puddle? Not the eggheads. No, it's always Al Vellucci and the ordinary working people who are left alone to wipe themselves off.
Whence this heat? Scientists at Harvard had voiced a desire to build an on-campus containment facility for doing recombinant work in strict accordance with the new NIH guidelines. But, seeing his chance and backed by a left-wing Harvard-MIT cabal with its own anti-DNA agenda, Vellucci managed to push through a several months' ban on all recombinant DNA research in Cambridge. The result was a brief but pronounced local brain drain, as Harvard and MIT biologists headed off to less politically charged climes. Vellucci, meanwhile, began to enjoy his newfound prominence as society's scientific watchdog. In 1977 he would write to the president of the National Academy of Sciences:
In today's edition of the Boston Herald American, a Hearst Publication, there are two reports which concern me greatly. In Dover, MA, a "strange, orange-eyed creature" was sighted and in Hollis, New Hampshire, a man and his two sons were confronted by a "hairy, nine foot creature."
I would respectfully ask that your prestigious institution investigate these findings. I would hope as well that you might check to see whether or not these "strange creatures" (should they in fact exist), are in any way connected to recombinant DNA experiments taking place in the New England area.
Though much debated, attempts to enact national legislation regulating recombinant DNA experiments fortunately never came to fruition. Senator Ted Kennedy of Massachusetts entered the fray early on, holding a Senate hearing just a month after Asilomar. In 1976, he wrote President Ford to advise that the federal government should control industrial as well as academic DNA research. In March of '77,1 testified before a hearing of the California state legislature. Governor Jerry Brown was in attendance, and so I had the occasion to advise him in person that it would be a mistake to consider any legislative action except in the event of unexplained illnesses among the scientists at Stanford. If those actually handling recombinant DNA remained perfectly healthy, the public would be better served if lawmakers focused on more evident dangers to public health, like bike riding.
As more and more experiments were performed, whether under NIH guidelines or under those imposed by regulators in other countries, it became more and more apparent that recombinant DNA procedures were not creating Frankenbugs (much less – pace Mr. Vellucci – "strange orange-eyed creatures"). By 1978 I could write, "Compared to almost any other object that starts with the letter D, DNA is very safe indeed. Far better to worry about daggers, dynamite, dogs, dieldrin, dioxin, or drunken drivers than to draw up Rube Goldberg schemes on how our laboratory-made DNA will lead to the extinction of the human race."
Later that year, in Washington, D.C., the Recombinant DNA Advisory Committee (RAC) of the NIH proposed much less restrictive guidelines that would permit most recombinant work – including tumor virus DNA research – to go forward. And in 1979, Joseph Califano, Secretary of Health, Education, and Welfare, approved the changes, thus ending a period of pointless stagnation for mammalian cancer research.
In practical terms, the outcome of the Asilomar consensus was ultimately nothing more than five sad years of delay in important research, and five frustrating years of disruption in the careers of many young scientists.
As the 1970s ended, the issues raised by Cohen and Boyer's original experiments turned gradually into non-issues. We had been forced to take an unprofitable detour, but at least it showed that molecular scientists wanted to be socially responsible.
Molecular biology during the second half of the 1970s, however, was not completely derailed by politics; these years did in fact see a number of important advances, most of them building upon the still controversial Boyer-Cohen molecular cloning technology.
The most significant breakthrough was the invention of methods for reading the sequence of DNA. Sequencing depends on having a large quantity of the particular stretch of DNA that you are interested in, so it was not feasible – except in the case of small viral DNA – until cloning technologies had been developed. As we have seen, cloning, in essence, involves inserting the desired piece of DNA into a plasmid, which is then itself inserted into a bacterium. The bacteria, allowed to divide and grow, will then produce a vast number of copies of the DNA fragment. Once harvested from the bacteria, this large quantity of the DNA fragment is then ripe for sequencing.
Two sequencing techniques were developed simultaneously, one by Wally Gilbert in Cambridge, Massachusetts (Harvard), and the other by Fred Sanger in Cambridge, England (see Plate 34). Gilbert's interest in sequencing DNA stemmed from his having isolated the repressor protein in the E. coli beta-galactosidase gene regulation system. As we have seen, he had shown that the repressor binds to the DNA close to the gene, preventing its transcription into RNA chains. Now he wanted to know the sequence of that DNA region. A fortuitous meeting with the brilliant Soviet chemist Andrei Mirzabekov suggested to Gilbert a way – using certain potent combinations of chemicals – to break DNA chains at just the desired, base-specific sites.
As a high-school senior in Washington, D.C., Gilbert used to cut class to read up on physics at the Library of Congress. He was then pursuing the Holy Grail of all high-school science prodigies: a prize in the Westinghouse Talent Search.* He duly won his prize in 1949. (Years later, in 1980, he would receive a call from the Swedish Academy in Stockholm, adding to the statistical evidence that winning the Westinghouse is one of the best predictors of a future Nobel.)
* In 1998, as the Old Economy gave way to the New, the honor was renamed the Intel Prize.
Gilbert stuck with physics as an undergraduate and graduate student, and a year after I arrived at Harvard in 1956 he joined the physics faculty. But once I got him interested in my lab's work on RNA, he abandoned his field for mine. Thoughtful and unrelenting, Gilbert has ever since been at the forefront of molecular biology.
Of the two sequencing methods, however, it is Sanger's that has better withstood the test of time. Some of the DNA-breaking chemicals required by G
ilbert's are difficult to work with; given half a chance, they will start breaking up the researcher's own DNA. Sanger's method, on the other hand, uses the same enzyme that copies DNA naturally in cells, DNA polymerase. His trick involves making the copy out of base pairs that have been slightly altered. Instead of using only the normal "deoxy" bases (As, Ts, Gs, and Cs) found naturally in DNA (deoxyribonucleic acid), Sanger also added some so-called "dideoxy bases." Dideoxy bases have a peculiar property: DNA polymerase will happily incorporate them into the growing DNA chain (i.e., the copy being assembled as the complement of the template strand), but it cannot then add any further bases to the chain. In other words, the duplicate chain cannot be extended beyond a dideoxy base.
Imagine a template strand whose sequence is GGCCTAGTA. There are many, many copies of that strand in the experiment. Now imagine that the strand is being copied using DNA polymerase, in the presence of a mixture of normal A, T, G, and C plus some dideoxy A. The enzyme will copy along, adding first a C (to correspond to the initial G), then another C, then a G, and another G. But when the enzyme reaches the first T, there are two possibilities: either it can add a normal A to the growing chain, or it can add a dideoxy A. If it picks up a dideoxy A, then the strand can grow no further, and the result is a short chain that ends in a dideoxy A (ddA): CCGGddA. If it happens to add a normal A, however, then DNA polymerase can continue adding bases: T, C, etc. The next chance for a dideoxy "stop" of this kind will not come until the enzyme reaches the next T. Here again it may add either a normal A or a ddA. If it adds a ddA, the result is another truncated chain, though a slightly longer one: this chain has a sequence of CCGGATCddA. And so it goes every time the enzyme encounters a T (i.e., has occasion to add an A to the chain); if by chance it selects a normal A, the chain continues, but in the case of a ddA the chain terminates there.
Where does this leave us? At the end of this experiment, we have a whole slew of chains of varying lengths copied from the template DNA; what do they all have in common? They all end with a ddA.
Now, imagine the same process carried out for each of the other three bases: in the case of T, for instance, we use a mix of normal A, T, G, and C plus ddT; the resultant molecules will be either CCGGAddT or CCGGATCAddT.
Having staged the reaction all four ways – once with ddA, once with ddT, once with ddG, and once with ddC – we have four sets of DNA chains: one consists of chains ending in ddA, one with chains ending with ddT, and so on. Now if we could only sort all these mini-chains according to their respective, slightly varying lengths, we could infer the sequence. How? A moment, please. First, let's see how we could do the sorting. We can place all the DNA fragments on a plate full of a special gel, and place the plate of gel in an electric field. In the pull of the electric field the DNA molecules will be forced to migrate through the gel, and the speed with which a particular mini-chain will travel is a function of its size: short chains travel faster than long ones. Within a fixed interval of time, the smallest mini-chain, in our case a simple ddC, will travel furthest; the next smallest, CddC, will travel a slightly shorter distance; and the next one, CCddG, a slightly shorter one still. Now Sanger's trick should be clear: by reading off the relative positions of all these mini-chains after a timed race through our gel, we can infer the sequence of our piece of DNA: first is a C, then another C, then a G, and so on.
In 1980, Sanger shared the Nobel Prize in Chemistry with Gilbert and with Paul Berg, who was recognized for his contribution to the development of the recombinant DNA technologies. (Inexplicably neither Stanley Cohen nor Herb Boyer has been so honored.)
For Sanger, this was his second Nobel.* He had received the chemistry prize in 1958 for inventing the method by which proteins are sequenced – that is, by which their amino acid sequence is determined – and applying it to human insulin. But there is absolutely no relation between Sanger's method for protein sequencing and the one he devised for sequencing DNA; neither technically nor imaginatively did the one give rise to the other. He invented both from scratch, and should perhaps be regarded as the presiding technical genius of the early history of molecular biology.
*As a double Nobelist, Sanger is in exalted company. Marie Curie received the prize in physics (1903) and then in chemistry (1911); John Bardeen received the physics prize twice, for the discovery of transistors (1956) and for superconductivity (1972); and Linus Pauling received the chemistry prize (1954) and the peace prize (1962).
Sanger is not what you might expect of a double Nobel laureate. Born to a Quaker family, he became a socialist and was a conscientious objector during the Second World War. More improbably, he does not advertise his achievements, preferring to keep the evidence of his Nobel honors in storage: "You get a nice gold medal, which is in the bank. And you get a certificate, which is in the loft." He has even turned down a knighthood: "A knighthood makes you different, doesn't it? And I don't want to be different." Having retired, Sanger is content these days to tend his garden outside Cambridge, though he still makes the occasional self-effacing and cheerful appearance at the Sanger Centre, the genome-sequencing facility near Cambridge that opened in 1993 (see Plates 31 & 32).
Sequencing would confirm one of the most remarkable findings of the 1970s. We already knew that genes were linear chains of As, Ts, Gs, and Cs, and that these bases were translated three at a time, in accordance with the genetic code, to create the linear chains of amino acids we call proteins. But remarkable research by Richard Roberts, Phil Sharp, and others revealed that, in many organisms, genes actually exist in pieces, with the vital coding DNA broken up by chunks of irrelevant DNA. Only once the messenger RNA has been transcribed is the mess sorted out by an "editing" process that eliminates the irrelevant parts. It would be as though this book contained occasional extraneous paragraphs, apparently tossed in at random, about baseball or the history of the Roman Empire. Wally Gilbert dubbed the intrusive sequences "introns" and the ones responsible for actual protein-coding (i.e., functionally part of the gene) he named "exons." It turns out that introns are principally a feature of sophisticated organisms; they do not appear in bacteria.
Some genes are extraordinarily intron-rich. For example, in humans, the gene for blood clotting factor VIII (which may be mutated in people with hemophilia) has twenty-five introns. Factor VIII is a large protein, some two thousand amino acids long, but the exons that code for it constitute a mere 4 percent of the total length of the gene. The remaining 96 percent of the gene is made up of introns.
Why, then, do introns exist? Obviously their presence vastly complicates cellular processes, since they always have to be edited out to form the messenger RNA; and that editing seems a tricky business, especially when you consider that a single error in excising an intron from the messenger RNA for, say, clotting factor VIII would likely result in a frameshift mutation that would render the resulting protein useless. One theory holds that these molecular intruders are merely vestigial, an evolutionary heirloom, left over from the early days of life on earth. Still it remains a much-debated issue how introns came to be and what if any use they may have in life's great code.
Once we became aware of the general nature of genes in eukaryotes (organisms whose cells contain a compartment, the nucleus, specialized for storing the genetic material; prokaryotes, such as bacteria, lack nuclei), a scientific gold rush was launched. Teams of eager scientists armed with the latest technology raced to be the first to isolate (clone) and characterize key genes. Among the earliest treasures to be found were genes in which mutations give rise to cancers in mammals. Once scientists had completed the DNA sequencing of several well-studied tumor viruses, SV40 for one, they could then pinpoint the exact cancer-causing genes. These genes were capable of transforming normal cells into cells with cancerlike properties, with for instance a propensity for the kind of uncontrolled growth and cell division that results in tumors. It was not long until molecular biologists began to isolate genes from human cancer cells, finally confirming that human cancer arises
because of changes at the DNA level and not from simple nongenetic accidents of growth, as had been supposed. We found genes that accelerate or promote cancer growth and we found genes that slow or inhibit it. Like an automobile, a cell, it seems, needs both an accelerator and a brake to function properly.
The treasure hunt for genes took over molecular biology. In 1981, Cold Spring Harbor Laboratory started an advanced summer course that taught gene-cloning techniques. Molecular Cloning, the lab manual that was developed out of this course, sold more than eighty thousand copies over the following three years. The first phase of the DNA revolution (1953-72) – the early excitement that grew out of the discovery of the double helix and led to the genetic code – eventually involved some three thousand scientists. But the second phase, inaugurated by recombinant DNA and DNA sequencing technologies, would see those ranks swell a hundredfold in little more than a decade.
Dna: The Secret of Life Page 12