Conversations with Laureates
DNA Drama
Reprinted from Dartmouth Medicine, Fall 1993
Today, even high school biology students know the dramatic story behind Watson and Crick's theoretical discovery of the structure of DNA - the message-bearer of all life. Lesser known are a DMS graduate's biochemical experiments that led to the breaking of the DNA code. Here's a behind-the-scenes look at some of modern biology's most fascinating discoveries.
By Rosemary LunardiniRosemary Lunardini is the assistant editor of Dartmouth Medicine magazine. Among her sources of information for this feature were: The Eighth Day of Creation by Horace Freeland Judson, Simon and Schuster, 1979; "Historical Aspects of Protein Synthesis" by Paul Zamecnik, in Harvard Cancer Commission Publication #1549, 1979; "Multiple Roles of Oligonucleotides in the Regulation of Genetic Expression" by Paul Zamecnik and Sudhir Agrawal, in New Leads and Targets in Drug Research, Copenhagen, 1992; and "Breaking the Code," in the November 28, 1992, issue of The Economist. William Kinlaw, M.D., an associate professor of medicine at Dartmouth Medical School, also contributed some thoughtful suggestions on the topic.
This October, scientists from all over the world will meet in Chicago to celebrate the 40th anniversary of the greatest biological discovery of the century: the unveiling of the structure of the DNA molecule by James Watson and Francis Crick. Paul Zamecnik, M.D. - a DC '33 and DMS '34 - will be among those there. He'll be taking a longer view than many on the mid-century discoveries related to the role of DNA in protein synthesis, for Zamecnik remains - after nearly 60 very productive years - a major player in the drama about which Crick said, "The secret of life is out."
Several months earlier, when he is in Hanover for his 60th Dartmouth College reunion, Zamecnik is asked if he agrees with Crick's assessment. "Close to it, in a way," he says in his careful manner. At age 80, he still works full-time as a principal investigator at the Worcester Foundation for Experimental Biology in Shrewsbury, Mass. "Why don't I retire? Well, I'm better at this than I am at gardening or carpentry," he says.
These days he is focused on the antisense strand of DNA, which is the noncoding strand that serves as the template for RNA synthesis. He is hopeful that pieces of antisense DNA, integrated into specific parts of the genome of viruses and bacteria, will block their replication. Ever since 1986, when he and his NIH collaborators were the first to report an antisense approach to inhibiting the HIV-1 virus, his main target has been the virus that causes AIDS.
Making sense of antisense - explaining how the process works: Zamecnik describes how the "gene machine" behind him (top) is used to synthesize the antisense oligonucleotides that are used to target the messenger RNA of viruses like HIV.
Eventually, he purifies the products of the gene machine using a liquid chromatograph (center, in picture above). He also spends some time working under a protective hood (bottom), feeding cultures of the viruses, protozoa, or bacteria that are potential targets for antisense therapy.
It was 1954 when Zamecnik first heard about Watson and Crick and their double-stranded DNA molecule which, when unraveled, replicated its protein-building instructions by a code that remained unknown. At the time, Zamecnik was totally involved in protein synthesis - an even bigger and harder problem than DNA structure. He would later see, however, that the two problems were intimately connected.
He remembers the very first question about proteins he ever posed. It was 1938, and he was an intern at University Hospitals in his hometown of Cleveland. When an overweight patient died for no apparent reason and the autopsy produced inconclusive results, he observed: "She was fat where she should have some muscles and protein. How are proteins synthesized? I talked to all the pundits there, and no one knew. One fellow said, 'There's someone at the Rockefeller Institute studying protein synthesis.' That was Max Bergmann."
Zamecnik was predisposed to science early on, although none of his medical colleagues were headed that way and he had never personally known anyone who did research. What motivated Zamecnik was his own impatience with medical knowledge in those days. "There was nothing you could do for patients," he says outright. So he read up on the great Bergmann, only to be turned down when he tried to get a job in his lab. "I want a chemist, not a medical man," Bergmann told him. Fortunately, Zamecnik landed a fellowship in Denmark in 1939 to work with a leader in the field of protein chemistry, Kaj Linderstrøm-Lang, at the Carlsberg Laboratory.
"At that time, Scandinavian research was tops," Zamecnik says. Unlike in America - where he claims research was conducted as a hobby by the rich or by clinicians not fully occupied with patient care - "the people in Denmark did it full-time. I thought these fellows were disciplined and knew what they were doing." The war cut short his stay there, however, and he and his wife, Mary, returned to the States. With, as he puts it, "stardust on my shoulders" from working with the Scandinavian scientists, this time he got Max Bergmann to take him on at the Rockefeller Institute.
There were, interestingly for a budding scientist, two competing views of protein synthesis in the 1940s. Bergmann believed they were made by enzymes, in a reversal of the way they were destroyed. Zamecnik joined the bright young group about Bergmann but before long moved to Boston to do wartime research with Joseph Aub. "During that time a funny fellow arrived at the Mass General named Fritz Lipmann, who had come from Denmark and had a notion that Bergmann was wrong," Zamecnik recalls. Lipmann thought there was a whole new pathway in which the amino acids were energized.
A lifetime of work in the lab: Zamecnik (center) with Drs. Mahlon Hoagland and Mary Stephenson, with whom he worked on cell-free protein synthesis at Harvard's Huntington Laboratories, 1956. - Photo by John Gilbert.
"He said to me at lunch one day, 'You don't really think these Bergmann enzymes have anything to do with protein synthesis, do you?' Then I thought, 'When the war is over, we'll try to study protein synthesis.' Carbon-14 was a by-product of the atomic energy project, and a whisper about it came through just after the war was over. I thought, 'Let's get that and find out whether Lipmann or Bergmann is right.'"
By measuring radioactive carbon-14 in amino acids, Zamecnik found Lipmann was right. "You had to energize these amino acids so they would link themselves together to form peptide chains. But how do they do it? Well, we found that they probably made ATP, a chemical energizer. However, these experiments were carried out in slivers of living cells in rat livers. You couldn't tell what was going on inside. I thought, 'We've got to break these cells up and then put them back together again. If we're lucky, we'll find out what the principal components are.' Five years went by, and nothing came out of it."
A sabbatical at Cal Tech with Linus Pauling was not productive either. "I learned nothing about protein synthesis, but I did get a better appreciation of chemistry - although not a great one," Zamecnik says. Arriving back in his Boston lab, which was now downstairs from Lipmann's lab, Zamecnik decided to try again to break proteins into smaller components.
Although Lipmann had put forward the idea of an "energy-rich bond" in the formation of a peptide chain, and Zamecnik had proved he was right, Zamecnik also wanted to know how the sequence of amino acids was arranged on the peptide chains to make proteins. Ever since Fred Sanger had shown that the sequence of amino acids in insulin was extremely complex, the idea had been abroad that each protein might have a unique structure.
This time, Zamecnik perfected his technique, which came to be known as the "cell-free system." It is described in The Eighth Day of Creation, an account of the 20th-century revolution in biology written by Horace Freeland Judson:
In the early fifties, a number of people were trying to make protein synthesis happen outside the cell, so that the process could be analyzed and played with. Here, Zamecnik's lab led. The idea was to put various combinations of cellular components and juices together, without the presence of living cells, and then to add labelled amino acids to see if the combination would link up some proteins. Such biochemical cocktails for protein synthesis in the test tube were called the cell-free system.
Zamecnik and his colleagues ground up cells with an abrasive, and threw down the grit and cell walls in a centrifuge at low speed. They tried E. coli, but after months of work found that they couldn't reliably get rid of whole cells. They turned to rat liver, minced and gently homogenized. After centrifuging that, they were left with a liquid containing all sorts of things - particles, nuclei, fragments of endoplasmic reticulum and other cellular structures, DNA, RNA, many different enzymes, and much else that was unknown or poorly known. . . . Effects could be followed in great detail when one or another item was labelled with radioactive isotopes. The flexibility and multiplicity of the possible experiments was in principle enormous.
Development, refinement, precise tuning of the cell-free system for protein synthesis was the chief technical advance of straight biochemistry in the decade of the fifties. . . . But even the simplest aim of getting a good and reproducible level of incorporation of amino acids into protein, without complete cells, only began to be possible in Zamecnik's lab in 1953 and '54.
The discoveries that came directly out of Zamecnik's lab or that flourished by using his cell-free system are like warp threads running the length of a history of the times. Zamecnik's first major discovery was to locate the site of protein synthesis. It occurred on microsomal particles - later to be called ribosomes. He thought of them as templates on which the proteins were made.
At that point, Dr. Mahlon Hoagland - who had most recently worked in Lipmann's lab and who later, in the 1960s, chaired DMS's biochemistry department - joined Zamecnik to help him find out where and how ATP provides energy for protein formation. "Contrary to our expectations that it should happen on the ribosomes, where the lineup of the amino acids had to occur if one were to have a distinct peptide sequence characteristic of any one protein, Mahlon found that the interaction occurred by way of ATP being broken down by an enzyme."
However, the energizing of the amino acids was just the first step in protein synthesis that the two colleagues were witnessing. The amino acids would still be assembled on the ribosomes. Elsewhere in the world, scientists were paying attention to this information. Horace Judson, in his chronicle, wrote of Zamecnik's pinpointing of the ribosome as the site of protein synthesis: "Crick found that definitive."
In 1953, Zamecnik missed a little article that came out in Nature on the structure of DNA. It was authored by two people he had never heard of (Watson and Crick), and it involved the use of x-ray crystallography (a technique he did not follow closely). Of course, nucleic acids were an ingredient in his cell-free solution and he knew that DNA was responsible for transmitting specific directions of inheritance.
"In 1954, I called my friend Paul Doty of Harvard, who was the high guru on DNA and RNA structure at that time. I said, 'Paul, what does the DNA do and how does it transmit the message to the sequence of the protein?' Paul Doty said, 'I happen to have a young fellow visiting me now. His name is James Watson, and if you'd like him to come over and talk to you, I'm sure he'd be glad to.'"
The meeting of the young Watson and the 40-year-old Zamecnik epitomized the apparently at-odds nature of two scientific tracks and two kinds of scientists. "James Watson came in with big eyes and looked very immature - like a high school student," Zamecnik recalls. "He had a wire model of DNA, and I learned for the first time of their double helix. It had been published nearly a year before, but I didn't hear of it. He set it up, and we looked at it."
Zamecnik was impressed by the model of the structure but began to question Watson - who with Crick was already becoming famous, at least in the small world of DNA research, for their bold and stunning theory. They were biologists, but they talked like physicists, with their abstractions about structure. Zamecnik was more attuned to the generation of classical biochemists like Lipmann, who sought sources of energy and identified materials. He asked Watson: "Where was protein made in all this? Where was DNA made? How did this complicated double helix unwind to reproduce itself?"
"He said, 'Gee, I have no idea.' So here he had this beautiful structure of DNA, which I accepted immediately, and we had proteins being made in our cell-free system, and there was a gap in between. How did the message of the gene get into making the sequence of the protein? There must be some kind of translation."
Shortly thereafter, Zamecnik began to pay more heed to the nucleic acids in his biochemical cocktail, and Watson, Crick, and their colleagues - in Zamecnik's words - "pricked up their ears and began to follow our lab very carefully."
A graphic representation of the concept behind transfer RNA: A missing translation piece identified by Zamecnik in 1957, transfer RNA binds to an amino acid, then "reads" the codon for that amino acid; the codons are carried by messenger RNA from DNA. In this way, amino acids are assembled into proteins. Today, Zamecnik calls tRNA "antisense DNA" and uses it to stop the growth of the peptide chain in viruses like HIV.
The next major development for Zamecnik would be the discovery of transfer RNA in 1956. RNA was a nucleic acid similar to DNA; it was also abundant in Zamecnik's broken-down cellular mix. In an experiment with Hoagland and Mary Stephenson in his lab, he found that both the ATP and the amino acid attached to some RNA. "There was a fraction of RNA which didn't spin down the way the usual RNA and DNA did, but remained in the supernatant with the soluble enzymes. It was known as soluble RNA. It was generally thought to be junk RNA - fragments here and there. It had no known function. . . . Sure enough, we found amino acid activation reached an intermediate step by way of this soluble RNA. . . . So you'd have to say we discovered transfer RNA, in which an amino acid was attached to a piece of RNA. It was there from its first activation as an amino acid by ATP." The lab's discovery "was a missing translation piece," adds Zamecnik. "I felt instantly, this is a translation piece between the code, which is present in DNA, and the sequencing of protein. But I didn't know how it worked."
Zamecnik says he only recently learned from Watson that Crick had written a little note to the "RNA Tie Club" in 1954 suggesting that a nucleotide - a chemical base on the DNA chain - might be a translation piece between DNA and protein. Zamecnik was not a member of the RNA Tie Club; he was an experimentalist, while the Tie Club was made up of theorists who gave themselves code names of amino acids as they exchanged ideas on how RNA might decode DNA. He first heard of Crick's suggestion in 1956, shortly after a Gordon Conference - a high-level, informal summer gathering of scientists - where Crick was later reported to have discussed his idea over sandwiches and beer one evening. "I didn't hear it. Mahlon didn't hear it," Zamecnik says of that evening. "But in a very circuitous way in the next few weeks, someone told us about it. By the time I heard it, third-hand, we had our sights on transfer RNA. So we said, 'Hey, we've got our fingers on exactly what he's talking about."
Later, it dawned on them that there were two functions to this transfer RNA. First, it had to recognize the enzyme that Hoagland had found activated the amino acids. Then it had to be recognized by the amino acids. "It's like going into a theater," Zamecnik says. "You get a ticket and from there on your face plays no role in the selection of your seat. Your ticket does. So the transfer RNA was the ticket to the sequence of the protein."
There was still a lot of RNA about, and how it all functioned had still not been pinned down. While Zamecnik and most other researchers first thought the RNA on the ribosome directed the sequencing of proteins, the concept of messenger RNA was now put forward by Jacques Monod and François Jacob at the Institut Pasteur. This RNA, they said, acted as a messenger by bringing the DNA's message to an all-purpose, non-specific ribosome. Crick characterized it as "a reading head."
"At that point, I felt like an observer on Mont-Saint-Michel when the tide begins to sweep past you," Zamecnik says. "A transient piece of RNA, which was made very quickly on demand by instructions from above from DNA and which disappeared rapidly - this messenger RNA - was responsible for putting the RNA, which was to be coded, on the ribosomes. Having them line up in the right way so that the piece of protein could be made very quickly . . . that was a big jump."
Meanwhile, Zamecnik's lab was working very hard on a bacterial cell-free system. Using bacteria to study the components of the cell would be much better than using rat liver; an experimenter in the lab cannot change the instructions in the cells of rat liver, whereas bacteria grow rapidly and can be more easily manipulated for experiments.
In 1960, after three years of work, postdoctoral fellow Robert Lamborg produced in Zamecnik's lab at Mass General a bacterial system in which there was no contamination of results by whole live cells. Even before this new technique was published, Zamecnik shared the bacterial cell-free system, which used E. coli, with his Harvard neighbors Jim Watson and Alfred Tissieres. Tissieres began to visit Zamecnik's lab. Within a year, reports of this brand-new, still-unpublished, bacterial cell-free system had spread along the scientific grapevine. Very quickly, it was used in the discovery of the next logical step - arguably the second most important biological discovery of the century: the breaking of the DNA code.
In the NIH lab of a young, then-unknown scientist, Marshall Nirenberg, a visiting German scientist named Johann Matthaei mixed the actual brew that deciphered the first "word" of the code. Looking back recently on this historic experiment, The Economist wrote: "His test-tube contained ribosomes, tiny particles obtained from the centrifuged and purified debris of a few million dead bacteria." This "brew" was straight from Zamecnik's lab.
"We gave the manuscript to Watson and Tissieres, and they took off from there. It wasn't their fault," Zamecnik says of the way his new technique was so quickly applied by others. "I would have to say we should have been a little more careful and waited until the whole thing was published. They made this bacterial system work better. Nirenberg paid attention to what Tissieres was doing, and then he used the cell-free system and had the courage to see whether he could find a crack in the code that way. I wouldn't have done that," Zamecnik admits. "I thought, 'You're trying to attach wings with paraffin and fly to the moon. We ought to do this in a more careful, sequential way.'"
Zamecnik has been questioned over the years as to why he did not pursue this next step, which initiated the decoding of DNA. He has answered that he was too close to the idea that the RNA on the ribosomes provided the specific instructions of the gene. He had had good reasons for thinking that way, because the peptide chain grew on the ribosomes (as he had proved) and because the ribosomes had plentiful RNA. The two in association might provide the primary code for building proteins. The fault in this view was in thinking of the ribosomes as specialized tools, each one designed to make a specific protein by translating a particular gene, whereas Nirenberg and Matthaei designed their code-cracking experiment as if the ribosomes were general, all-purpose reading machines.
"For a time, it was considered possible that ribosomal RNA itself provided the genetic message," Zamecnik wrote in a 1984 review. "The logic of one year, however, has a way of becoming the naiveté of the next.
"In its early days, messenger RNA was an intangible substance, from the biochemist's point of view. Following the development of a bacterial cell-free system, it became easier to demonstrate that the genetic message could change without degradation and resynthesis of the ribosomal particle itself. The evidence for messenger RNA plus the cell-free bacterial system made a breathtaking experiment possible," he wrote.
"We had reprints turning yellow for five years," Zamecnik rues: When he published a paper in 1978 on the sequencing of oligonucleotides, Zamecnik thought the discovery would "make a stir" in the scientific world. But it took quite a while before the work was recognized for its role in the protein synthesis puzzle that has occupied him for more than 60 years.
Within five years, the rest of the code had been deciphered, as many other labs jumped into the race. Zamecnik's lab did not. He stuck to transfer RNA - trying to purify and sequence it. "The code-deciphering field had moved too fast. But in the end, so did transfer RNA sequencing," he told an assembly of scientists in 1979, after delivering a major address on the history of protein synthesis.
But Zamecnik would discover offshoots of his labors with the negative-stranded RNA. In 1978, his lab - some 20 years after his first major discoveries - observed that sequences of more than two nucleotides, called oligonucleotides, sometimes got into cells. "They weren't supposed to," he says. "We published this paper, and it seemed to me it was nifty. It would make a stir. Nothing. We had reprints turning yellow for five years."
However, this was the beginning of a new direction to his own work, and it eventually led to a whole new branch of research that last year produced 700 papers - more than Zamecnik could find time to read. He had surmised that if oligonucleotides contained the reverse sequence to that of a messenger RNA encoding an enzyme necessary to replicate a virus, they could be delivered into cells infected with the virus. They could hybridize with the target and block the virus from replicating.
To use them therapeutically it would be necessary to sequence RNA or DNA from viruses and to synthesize nucleotides of sufficient length so that they would hybridize well with a segment of viral RNA or DNA. For example, if one knew the primary sequence of a negative-stranded RNA virus - influenza, for example - "it would be possible to synthesize and test its oligonucleotide complement as a possible virus inhibitor," he wrote with Mary Stephenson in 1978.
Several things converged in the early 1980s that helped him to investigate this possibility. There were better ways of synthesizing oligonucleotides; powerful advances in DNA sequencing; sequences available from viruses and parts of the human genome; and ways of inserting rather large pieces of the genome into specific sites.
"It really goes back to transfer RNA, which you can call 'antisense' DNA," he says. The strand of DNA that carries the message from RNA to the protein is the 'sense' strand. And the antisense strand, which is hybridized to the sense strand, he explains, "was supposed to be silent, to do nothing but serve as the photographic negative so you could make another positive. But I thought for a long time, and so did other people, that it must be doing other things. You don't waste that much information."
In 1992, he might have been summing up his own long affair with transfer RNA when he wrote: "So it was that transfer RNA appeared next in line after the double helix, as a separate, unexpected finding, depending for its appearance on its predecessor. This remarkable macromolecule provided a mechanism for conversion of the language of the gene into that of the protein.
"It was further exciting to find in transfer RNA the first example of a negative strand sequence used for a purpose other than as a photographic negative. Transfer RNA acts in a positive metabolic way by specifying the sequence of a growing peptide chain and by activating its polymerization. At the same time, it performs an inhibitory antisense function, stopping the growth of the peptide chain. . . . It further serves as a primer for retroviruses, once again acting in a positive fashion even though an antisense strand."
In his own mind, as a physician-turned-scientist, Zamecnik feels the earlier discoveries of DNA's role in protein synthesis left a gap between pure knowledge and its application in medicine. Making use of antisense DNA is, he says, "a rational way to approach disease." It fits into the category of chemotherapeutics, and thus far Zamecnik's lab has targeted influenza, malaria, and AIDS (the latter with the NIH's Robert Gallo, codiscoverer of the AIDS virus).
With a piece of antisense DNA, he explains, "You try to target some piece of the genome of a virus which is invariant and doesn't change from one virus to the next one. That's part of the problem in making an AIDS vaccine. . . . With the antisense approach, we look at the DNA from San Francisco, from our area, from Africa, and select the pieces which are invariant and try to target them."
In the last year, his research group has reported inhibition of the HIV-1 virus, without toxicity to T-cells, in tissue cultures for at least 84 days. Their results suggest that an in vivo therapy with antisense pieces would be best achieved with an initial high dose, followed by lower maintenance doses. This antisense therapy will soon enter two clinical trials - the first sponsored by the French government and the second by the National Institutes for Allergy and Infectious Diseases in this country.
Over the years, Zamecnik has received many awards for his pathbreaking research: Five honorary doctorates in science - from the University of Utrecht, Columbia, Harvard, Dartmouth, and Roger Williams College. The Borden Award in Medical Sciences. The Passano Award. And, in 1991, the Presidential Medal of Science.
The nation's most prestigious scientific honor: In 1991, Zamecnik was presented with the National Medal of Science by President George Bush. The citation accompanying it reads: "To Paul Zamecnik, for his pioneering research of protein biosynthesis, opening the door to biochemical attack and paving the way for dissection of the genetic code; and for introducing the concept and method of 'antisense DNA' as an approach to viral gene inhibition and chemotherapy".
Zamecnik is just as pleased with his work on antisense DNA as with his earlier work in the heady days of protein synthesis. Should his 1978 paper, yellowed with age before anyone took notice of it, be considered the first recognition of the potential of antisense DNA? "Well, I don't know," he says. "Let's just say it was pioneer work, and it had its roots back in the era of transfer RNA."
At a Dartmouth Medical School luncheon during his recent visit to campus, sitting with classmates who practiced clinical medicine all their lives, Zamecnik heard Dean Andrew Wallace describe the new joint M.D./Ph.D. program at Dartmouth Medical School - something that might have suited him had it been around when he was in medical school. "It's fortunate the organic chemists I have to associate with are willing to put up with my medical student-size organic chemistry," he says. "I wish I'd had not a Ph.D. per se, but I wish I had known theoretical organic chemistry better. However, scientists can put on blinders and not see diseases. They don't have a background that points their lantern in the direction of a variety of diseases."
Paul Zamecnik, for all his years in the lab, has not forgotten where his own lantern points - towards the discovery of rational ways to approach disease.