Jacob too was involved in the war effort, having escaped to Britain and joined General de Gaulle's Free French Army. He served in North Africa and participated in the D-day landings. Shortly thereafter, he was nearly killed by a bomb; twenty pieces of shrapnel were removed, but he retains to this day another eighty. Because his arm was damaged, his injuries ended his ambition to be a surgeon, and, inspired like so many of our generation by Schrödinger's What Is Life?, he drifted toward biology. His attempts to join Monod's research group were, however, repeatedly rebuffed. But after seven or eight tries, by Jacob's own count, Monod's boss, the microbiologist Andre Lwoff, caved in in June 1950 (see Plate 24):
Without giving me a chance to explain anew my wishes, my ignorance, my eagerness, [Lwoff] announced, "You know, we have discovered the induction of the prophage!" [i.e., how to activate bacteriophage DNA that has been incorporated into the host bacterium's DNA].
I said, "Oh!" putting into it all the admiration I could and thinking to myself, "What the devil is a prophage?"
Then he asked, "Would it interest you to work on phage?" I stammered out that that was exactly what I had hoped. "Good; come along on the first of September."
Jacob apparently went straight from the interview to a bookshop to find a dictionary that might tell him what he had just committed himself to.
Despite its inauspicious beginnings, the Jacob-Monod collaboration produced science of the very highest caliber. They tackled the gene-switching problem in E. coli, the familiar intestinal bacterium, focusing on its ability to make use of lactose, a kind of sugar. In order to digest lactose, the bacterium produces an enzyme called beta-galactosidase, which breaks the nutrient into two subunits, simpler sugars called galactose and glucose. When lactose is absent in the bacterial medium, the cell produces no beta-galactosidase; when, however, lactose is introduced, the cell starts to produce the enzyme. Concluding that it is the presence of lactose that induces the production of beta-galactosidase, Jacob and Monod set about discovering how that induction occurs.
In a series of elegant experiments, they found evidence of a "repressor" molecule that, in the absence of lactose, prevents the transcription of the beta-galactosidase gene. When, however, lactose is present, it binds to the repressor, thereby keeping it from blocking the transcription; thus the presence of lactose enables the transcription of the gene. In fact, Jacob and Monod found that lactose metabolism is coordinately controlled: it is not simply a matter of one gene being switched on or off at a given time. Other genes participate in digesting lactose, and the single repressor system serves to regulate all of them. While E. coli is a relatively simple system in which to investigate gene-switching, subsequent work on more complicated organisms, including humans, has revealed that the same basic principles apply across the board.
Jacob and Monod obtained their results by studying mutant strains of E. coli. They had no direct evidence of a repressor molecule: its existence was merely a logical inference from their solution to the genetic puzzle. Their ideas were not validated in the molecular realm until the late sixties, when Walter (Wally) Gilbert and Benno Müller-Hill at Harvard set out to isolate and analyze the repressor molecule itself. Jacob and Monod had only predicted its existence; Gilbert and Müller-Hill actually found it. Because the repressor is normally present only in tiny amounts, just a few molecules per cell, gathering a sample large enough to analyze proved technically challenging. But they got it in the end. At the same time, Mark Ptashne, working down the hall in another lab, managed to isolate and characterize another repressor molecule, this one in a bacteriophage gene-switching system. Repressor molecules turn out to be proteins that can bind to DNA. In the absence of lactose, then, that is exactly what the beta-galactosidase repressor does: by binding to a site on the E. coli DNA close to the point at which transcription of the beta-galactosidase gene starts, the repressor prevents the enzyme that produces messenger RNA from the gene from doing its job. When, however, lactose is introduced, that sugar binds to the repressor, preventing it from occupying the site on the DNA molecule close to the beta-galactosidase gene; transcription is then free to proceed.
The characterization of the repressor molecule completed a loop in our understanding of the molecular processes underpinning life. We knew that DNA produces protein via RNA; now we also knew that protein could interact directly with DNA, in the form of DNA-binding proteins, to regulate a gene's activity.
The discovery of the central role of RNA in the cell raised an interesting (and long-unanswered) question: why does the information in DNA need to go through an RNA intermediate before it can be translated into a polypeptide sequence? Shortly after the genetic code was worked out, Francis Crick proposed a solution to this paradox, suggesting that RNA predated DNA. He imagined RNA to have been the first genetic molecule, at a time when life was RNA-based: there would have been an "RNA world" prior the familiar "DNA world" of today (and of the past few billion years). Crick imagined that the different chemistry of RNA (based on its possession of the sugar ribose in its backbone, rather than the deoxyribose of DNA) might endow it with enzymatic properties that would permit it to catalyze its own self-replication.
Crick argued that DNA had to be a later development, probably in response to the relative instability of RNA molecules, which degrade and mutate much more easily than DNA molecules. If you want a good stable, long-term storage molecule for genetic data, then DNA is a much better bet than RNA.
Crick's ideas about an RNA world preceding the DNA one went largely unnoticed until 1983. That's when Tom Cech at the University of Colorado and Sidney Altman at Yale independently showed that RNA molecules do indeed have catalytic properties, a discovery that earned them the Nobel Prize in Chemistry in 1989. Even more compelling evidence of a pre-DNA RNA world came a decade later, when Harry Noller at the University of California, Santa Cruz, showed that the formation of peptide bonds, which link amino acids together in proteins, is not catalyzed by any of the sixty different proteins found associated with the ribosome, the site of protein synthesis. Instead, peptide bond formation is catalyzed by RNA. He arrived at this conclusion by stripping away all the proteins from the ribosome and finding that it was still capable of forming peptide bonds.
Exquisitely detailed analysis of the 3-D structure of the ribosome by Noller and others shows why: the proteins are scattered over the surface, far from the scene of action at the heart of the ribosome (see Plate 25).
These discoveries inadvertently resolved the chicken-and-egg problem of the origin of life. The prevailing assumption that the original life-form consisted of a DNA molecule posed an inescapable contradiction: DNA cannot assemble itself; it requires proteins to do so. Which came first? Proteins, which have no known means of duplicating information, or DNA, which can duplicate information but only in the presence of proteins? The problem was insoluble: you cannot, we thought, have DNA without proteins, and you cannot have proteins without DNA.
RNA, however, being a DNA equivalent (it can store and replicate genetic information) as well as a protein equivalent (it can catalyze critical chemical reactions) offers an answer. In fact, in the "RNA world" the chicken-and-egg problem simply disappears. RNA is both the chicken and the egg.
RNA is an evolutionary heirloom. Once natural selection has solved a problem, it tends to stick with that solution, in effect following the maxim "If it ain't broke, don't fix it." In other words, in the absence of selective pressure to change, cellular systems do not innovate and so bear many imprints of the evolutionary past. A process may be carried out in a certain way simply because it first evolved that way, not because that is absolutely the best and most efficient way.
Molecular biology had come a long way in its first twenty years after the discovery of the double helix. We understood the basic machinery of life, and we even had a grasp on how genes are regulated. But all we had been doing so far was observing; we were molecular naturalists for whom the rain forest was the cell – all we could do was describe what
was there. The time had come to become proactive. Enough observation: we were beckoned by the prospect of intervention, of manipulating living things. The advent of recombinant DNA technologies, and with them the ability to tailor DNA molecules, would make all this possible.
CHAPTER FOUR
PLAYING GOD:
CUSTOMIZED DNA MOLECULES
DNA molecules are immensely long. Only one continuous DNA double helix is present in any given chromosome. Popular commentators like to evoke the vastness of these molecules through comparisons to the number of entries in the New York City phone book or the length of the River Danube. Such comparisons don't help me – I have no sense of how many phone numbers there are in New York City, and mention of the Danube more readily suggests a Strauss waltz than any sense of linear distance.
Except for the sex chromosomes, X and Y, the human chromosomes are numbered according to size. Chromosome 1 is the largest and chromosomes 21 and 22 are the smallest. In chromosome 1 there resides 8 percent of each cell's total DNA, about a quarter of a billion base pairs. Chromosomes 21 and 22 contain some 40 and 45 million base pairs respectively. Even the smallest DNA molecules, those from small viruses, have no fewer than several thousand base pairs.
The great size of DNA molecules posed a big problem in the early days of molecular biology. To come to grips with a particular gene – a particular stretch of DNA – we would have to devise some way of isolating it from all the rest of the DNA that sprawled around it in either direction. But it was not only a matter of isolating the gene; we also needed some way of "amplifying" it: obtaining a large enough sample of it to work with. In essence we needed a molecular editing system: a pair of molecular scissors that could cut the DNA text into manageable sections; a kind of molecular glue pot that would allow us to manipulate those pieces; and finally a molecular duplicating machine to amplify the pieces that we had cut out and isolated. We wanted to do the equivalent of what a word processor can now achieve: to cut, paste, and copy DNA.
Developing the basic tools to perform these procedures seemed a tall order even after we cracked the genetic code. A number of discoveries made in the late sixties and early seventies, however, serendipitously came together in 1973 to give us so-called "recombinant DNA" technology – the capacity to edit DNA. This was no ordinary advance in lab techniques. Scientists were suddenly able to tailor DNA molecules, creating ones that had never before been seen in nature. We could "play God" with the molecular underpinning of all of life. This was an unsettling idea to many people. Jeremy Rifkin, an alarmist for whom every new genetic technology has about it the whiff of Dr. Frankenstein's monster, had it right when he remarked that recombinant DNA "rivaled the importance of the discovery of fire itself."
Arthur Kornberg was the first to "make life" in a test tube. In the 1950s, as we have seen, he discovered DNA polymerase, the enzyme that replicates DNA through the formation of a complementary copy from an unzipped "parent" strand. Later he would work with a form of viral DNA; he was ultimately able to induce the replication of all of the virus's 5,300 base pairs of DNA. But the product was not "alive"; though identical in DNA sequence to its parent, it was biologically inert. Something was missing. The missing ingredient would remain a mystery until 1967, when Martin Gellert at the National Institutes of Health and Bob Lehman at Stanford simultaneously identified it. This enzyme was named "ligase." Ligase made it possible to "glue" the ends of DNA molecules together.
Kornberg could replicate the viral DNA using DNA polymerase and, by adding ligase, join the two ends together so that the entire molecule formed a continuous loop, just as it did in the original virus. Now the "artificial" viral DNA behaved exactly as the natural one did: the virus normally multiplies in E. coli, and Rornberg's test-tube DNA molecule did just that. Using just a couple of enzymes, some basic chemical ingredients, and viral DNA from which to make the copy, Kornberg had made a biologically active molecule. The media reported that he had created life in a test tube, inspiring President Lyndon Johnson to hail the breakthrough as an "awesome achievement."
The contributions of Werner Arber in the 1960s to the development of recombinant DNA technology were less expected. Arber, a Swiss biochemist, was interested not in grand questions about the molecular basis of life but in a puzzling aspect of the natural history of viruses. He studied the process whereby some viral DNAs are broken down after insertion into bacterial host cells. Some, but not all (otherwise viruses could not reproduce), host cells recognized certain viral DNAs as foreign, and selectively attacked them. But how – and why? All DNA throughout the natural world is the same basic molecule, whether found in bacteria, viruses, plants, or animals. What kept the bacteria from attacking their own DNA even as they went after the virus's?
The first answer came from Arber's discovery of a new group of DNA-degrading enzymes, restriction enzymes. Their presence in bacterial cells restricts viral growth by cutting foreign DNA. This DNA-cutting is a sequence-specific reaction: a given enzyme will cut DNA only when it recognizes a particular sequence. EcoRl, one of the first restriction enzymes to be discovered, recognizes and cuts the specific sequence of bases GAATTC.
But why is it that bacteria do not end up cutting up their own DNA in every place where the sequence GAATTC appears? Here Arber made a second big discovery. While making the restriction enzyme that targets specific sequences, the bacterium also produces a second enzyme that chemically modifies those very same sequences in its own DNA wherever they may occur.* Modified GAATTC sequences present in the bacterial DNA will pass unrecognized by EcoRl, even as the enzyme goes its marauding way, snipping the sequence wherever it occurs in the viral DNA.
* The enzyme achieves this chemical modification by adding methyl groups, CH3, to the bases.
The next ingredient of the recombinant DNA revolution emerged from studies of antibiotic resistance in bacteria. During the sixties, it was discovered that many bacteria developed resistance to an antibiotic not in the standard way (through a mutation in the bacterial genome) but by the import of an otherwise extraneous piece of DNA, called a "plasmid." Plasmids are small loops of DNA that live within bacteria (see Plate 26) and are replicated and passed on, along with the rest of the bacterial genome, during cell division. Under certain circumstances plasmids may also be passed from bacterium to bacterium, allowing the recipient instantly to acquire a whole cassette of genetic information it did not receive "at birth." That information often encompasses the genes conferring antibiotic resistance. Natural selection imposed by antibiotics favors those bacterial cells that have the resistance factor (the plasmid) on board.
Stanley Cohen, at Stanford University, was a plasmid pioneer. Thanks to the encouragement of his high-school biology teacher, Cohen opted for a medical career. Upon graduation from medical school, his plans to practice internal medicine were shelved when the prospect of being drafted as an army doctor inspired him to accept a research position at the National Institutes of Health. He soon found that he preferred research over practicing medicine. His big breakthrough came in 1971, when he devised a method to induce E. coli bacterial cells to import plasmids from outside the cell. Cohen was, in effect, "transforming" the E. coli as Fred Griffith, forty years before, had converted strains of nonlethal pneumonia bacteria into lethal ones through the uptake of DNA. In Cohen's case, however, it was the plasmid, with its antibiotic resistance genes, that was taken up by a strain that had previously been susceptible to the antibiotic. The strain would remain resistant to the antibiotic over subsequent generations, with copies of the plasmid DNA passed along intact during every cell division.
By the early seventies, all the ingredients to make recombinant DNA were in place. First we could cut DNA molecules using restriction enzymes and isolate the sequences (genes) we were interested in; then, using ligase, we could "glue" that sequence into a plasmid (which would thus serve as a kind of floppy disk containing our desired sequence); finally, we could copy our piece of DNA by inserting that same plasmid floppy into a
bacterial cell. Ordinary bacterial cell division would take care of replicating the plasmid with our piece of DNA just as it would the cell's own inherited genetic materials. Thus, starting with a single plasmid transplanted into a single bacterial cell, bacterial reproduction could produce enormous quantities of our selected DNA sequence. As we let that cell reproduce and reproduce, ultimately to grow into a vast bacterial colony consisting of billions of bacteria, we would be simultaneously creating billions of copies of our piece of DNA. The colony was thus our DNA factory.
Dna: The Secret of Life Page 10
