Nobelist Berg Reflects On Recombinant DNA
The issues examined in this edition of C&EN concern potential applica tions of genetic engineering. How ever, the tools of genetic engineering originally were developed to assist basic research into molecular genetics. That basic research continues unabated, and its ongoing achievements are sometimes overshadowed by unrelated controversies and by the attention lavished on the biotechnology industry. Stanford University biochemist Paul Berg is a semi nal figure in recombinant DNA research. In the early 1970s Berg and coworkers created the first recombi nant DNA molecules. For that work, he shared the 1980 Nobel Prize for Chemistry with Walter Gilbert and Frederick Sanger, who were cited for their develop ment of another important tool of genetic engineering, a technique for the rapid sequencing of DNA molecules. Berg's work on what he calls genetic chemistry continues today. He and his coworkers use recombi nant DNA techniques to construct synthetic minichro mosomes, which are then introduced into cells to study processes such as genetic recombination, modification of defective sequences, and promoter function. Berg's laboratory also is involved in efforts to develop more sophisticated techniques for isolating specific mamma lian genes. Berg played a central role in the early recombinant DNA controversies. In 1971, Janet Mertz, then a student of Berg's, described to a meeting at Cold Spring Harbor Laboratory, Long Island, a proposed experi ment that would introduce a tumor virus chromosome into Escherichia coli. The concerns raised by many scientists over the safety of such an experiment prompted Berg to postpone it. By 1973, a number of experiments describing molecu lar cloning were presented at that year's Gordon Con ference on Nucleic Acids, prompting the now-famous letter from Maxine Singer and Dieter Soil, the cochairmen of the conference, to the National Acade my of Sciences president, who at that time was Philip Handler. That letter led to the formation of the Ν AS
Committee on Recombinant DNA, which Berg chaired. The recommendations of that committee resulted in a moratorium on several types of recombinant DNA experiments and, eventually, to the formation of the National Institutes of Health Recombinant DNA Advi sory Committee (RAC). Berg also organized the Asilomar Conference on Recombinant DNA Molecules held in February 1975. The recommendations of that conference led to the containment guidelines adopted by RAC for recombi nant DNA experiments. Subsequently, Berg worked to deflect legislation aimed at limiting or banning recombi nant DNA experiments and also worked to relax the RAC guidelines as experience with recombinant DNA eased scientists' concerns over the safety of the experiments. In a recent interview with C&EN San Francisco bureau head Rudy M. Baum, Berg discussed some of the major advances in molecular biology spawned by recombinant DNA, likely future research directions, and his views on past and present controversies relat ing to genetic engineering.
With respect to recombinant DNA technology, we hear mainly about commercial applications or basic research in universities that has potential applications. Yet the techniques were developed to probe genetic molecular biology, and that work continues today. What have been some of the major accomplishments of that effort? I think one should recognize that recombinant DNA technology did not emerge suddenly. Many of the basic approaches on which recombinant DNA relies already existed in the early 1970s, largely through work on the molecular biology of such organisms as E. August 13, 1984 C&EN
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Genetic Engineering Report f labeled gene A, gene B, but actual nucleotide se| quences of entire regions encompassing five to 10 S genes. I think that if anyone had said in 1970 that that would be accomplished or even feasible in our lifetimes, 1980 Nobel Prize he or she would have been laughed out of any meeting. One of the major discoveries that followed from the recognized cloning ability to isolate genes and analyze their structures and nucleotide was that genes from mammals are very different in structure from those of bacteria. Mammalian genes sequencing as the are far more complex, having intervening sequences, or introns, within the coding information. No one key advances that anticipated such a novel structure. opened the field; That discovery posed a major question of where the intervening sequences came from, what they do, and each alone would how they are removed from the RNA transcript to permit the synthesis of protein. Now we recognize have far less that one of the major problems of messenger RNA of an impact formation is splicing. Much of our present knowledge of this important reaction stems from the ability to isolate these genes, to identify the structures of intervening sequences, and, using molecular cloning, to devise approaches that permit an analysis of how intervening sequences are removed during the posttranscriptional phase of mRNA synthesis. As it turns out, splicing is not a simple process. coli and its bacteriophages. During the 1960s and early 1970s many investigators believed that if we Another important advance dependent on recombiunderstood the genetic structure of E. coli and its nant DNA techniques is the ability to make mutations phages, and their basic regulatory mechanisms, we at specific locations in isolated genes. So now we can would also understand the genetics of higher orgando what the chemists have done for a long, long time. isms. No one doubted that the regulatory machinery The chemical structure of DNA molecules can be of higher organisms would be a bit more complicated, c h a n g e d in very specific ways so that specific but the general feeling was that the differences would mutational alterations can be created at virtually any more likely be variations on the bacterial theme rathor every base one wants to study. Such specifically er than something completely novel. altered genes can then be used to assess the functional role of different parts of the gene. Generally, this However, because the genomes of higher organrequires an assay for gene function, and is performed isms are enormously more complex, not too many by reintroducing the genes back into cells to deterpeople were prepared to try to tackle these systems mine their expression. experimentally. Certainly, there was almost no prospect of isolating specific genes from eukaryote genomes and analyzing their structures. The ability to do these sorts of studies depended on Nevertheless, several groups were working on aniadvances in areas, such as sequencing, other than mal cells in culture with the expectation that eventualrecombinant DNA technology, didn't they? ly it would be possible to manipulate their genetic Of course. Isolating genes is only the beginning step structures in ways comparable to E. coli. The rein characterizing their structures and functions. But combinant DNA technology provided just such a ca- cloning provides one with a pure DNA segment, and pability. that made sequencing of genes much easier. Quite clearly, sequencing mixtures of DNA segments would What were the first sorts of experiments that were have been impossible. So, molecular cloning and DNA sequencing are at the core of recombinant DNA done? technology. Indeed, the 1980 Nobel Prize recognized The first efforts were aimed at dissecting complicated that cloning, which made possible the isolation of genomes to obtain bits and pieces of genomic DNA and to look at their structures to see if specific genes pure DNA segments, and nucleotide sequencing, which made it possible to characterize discrete DNA segments, could be identified. And, much as you would do with the bits and pieces of a jigsaw puzzle, a clear objective were the key advances that opened the field. Each was to reconstruct the original arrangement. That is alone would have had far less of an impact. Cloning of DNA molecules without being able to determine what researchers have now done. Such experiments have produced two quite extraor- their sequence would not have been able to provide the detailed chemical information about the DNA or dinary results. First, it is possible to isolate virtually any gene or genes from any organism. Second, it is gene structures. On the other hand, meaningful nucleopossible to determine the entire nucleotide sequence tide sequencing could not have provided useful information unless purified pieces of DNA were available of those genes and then to define their arrangement along the chromosome—not just maps with boxes for sequencing. 60
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These combined capabilities, cloning and sequencing, allow investigators to dissect and analyze extended regions of complex genomes and to define the location, arrangement, and structure of genes. For instance, if you want to study the gene that codes for insulin and the regulation of its expression, the process is nearly routine. The gene coding for the protein is first isolated, its sequence is analyzed to define its functional elements—the promoter, coding sequences, introns, and termination signals—and then methods are devel oped to reintroduce the gene into suitable cells so that its expression can be assayed. Then, the nowclassical approach is to modify the gene's sequence at specific locations to determine which elements of the sequence are important for its expression and regula tion. Ideally, one would like to know what every single nucleotide in a gene's sequence is needed for. What else has been discovered about eukaryotic genes? Beyond the important lesson that the genes of higher organisms are far more complex than anybody had imagined, most genes were found to have unusual arrangements relative to one another and to adjoining sequences. Eukaryote genes are not "nose to nose" in the chromosome as they are in bacteria. Generally there are large spaces between genes. The β-globinlike genes, for example, are arranged on one chromosome, but there are probably several thousand nucleotides between, say, the δ-globin gene and the /3-globin gene. The two α-globin genes are also separated by a few thousand nucleotide pairs. So, unlike £. coli or its phages, in which genes are arranged adjacent to one another or even overlap, eukaryote genes seem to be separated by substantial di&tances. What about the excess DNA between functional genes? How is that explained? A very large part of the DNA between genes consists of a variety of repeated sequences. Some are very short sequences and are repeated more than a million times. There are other kinds of repeated sequences that are repeated several hundred to a thousand times. Often the repeated sequences form families that are dispersed throughout the genome. In some cases, the sequences are tandemly repeated and clustered at spe cial places in the chromosomes. At present, we know very little about their function. People working in this field are studying the molecular anatomy of com plex genomes. The feeling is that understanding the anatomy fully may reveal answers as to the function of various sequence arrangements. Application of the recombinant DNA technique also helped establish that the arrangement of genes and repeated elements is not fixed. That is, certain DNA elements move from one place to another. In some cases these movements are programed and absolutely essential for normal function. In other cases, the rear rangement appears to be random and undirected. For example, in the formation of immunoglobulins, gene segments are rearranged during the develop ment of the B-cells to form the functional gene. This
rearrangement occurs only in B-cells and must occur properly for the cells to make an antibody. In summary, I think the thing most scientists work ing in this field are most excited about is the ability to isolate genes from any organism, determine their chemical structures, define their chromosomal organ ization, and study their structure-function relationships. The last aspect often depends on being able to put genes back into cells. Why is that so important? Putting genes back into the chromosomes of living cells allows us to study how these transplanted genes are expressed. Some researchers have used in-vitro systems to assay for the function of isolated genes, but those systems are much further behind in their development. Therefore, much of the emphasis is to develop methods for putting genes back into cells and more recently into whole animals. Isolated genes can be incorporated into vector systems that reintroduce the genes into specific cells of whole animals. One of the major challenges that researchers are trying to get at now is to determine to what extent the proper regulation of a gene is a function of its loca tion and its neighboring sequences. We need to know if putting a gene that is normally on chromosome 11 into, say, chromosome 9, will still permit it to be regulated properly. So far, it looks like it can be in some instances but, in many other cases, it behaves aberrantly. We've been talking mainly about genetic function per se. Recombinant DNA technology also has had a major impact on other areas of biological sciences such as the understanding of immunology, oncology, and development, hasn't it? Those all follow pretty much from what we have been talking about. For instance, David Hogness, one of my
A major challenge facing researchers is to determine to what extent the proper regulation of a gene is a function of its location and of its neighboring sequences
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Genetic Engineering Report colleagues at Stanford, is very much interested in the development of Drosophila. He has chosen to study a set of genes that controls the development of the fly's thorax. The first step involved the cloning of the entire region of the genome that controls that aspect of the fly's development. This region spans nearly 100,000 nucleotide pairs and contains all of the genet ic information needed for the program for developing the organism's thorax. Hogness and his students de v e l o p e d a t e c h n i q u e called w a l k i n g a l o n g t h e chromosome. It involves making gene banks of Dro sophila DNA, isolating those that are relevant to this set of genes, and defining the arrangement of all the cloned fragments from that region. Similar approaches are being used to study the de velopment of other parts of Drosophila, and other researchers are applying this same basic approach to study the development of the mouse. In all cases, the goal is to identify the genes that are involved in the developmental program, isolate them, sequence them, find the proteins that they code for, determine the function of those proteins, and discover the mecha nism of their regulation. The field of oncology has been revolutionized by recombinant DNA techniques. These studies have shown our own genes are causing our cancers. Many of the oncogenes have been identified, and the mutational alterations that cause them to produce can cer also have been established. The next step is to determine how the products of these genes trigger the normal cell into a cancer state. Recombinant DNA methods also have created a whole range of diagnostic capabilities that have very practical consequences. In essence, it is possible by examining the structure of a cell's DNA to determine if a particular gene has a normal or defective structure. That certainly has enormous implications for the pre natal diagnosis of genetic disease. What is, so to speak, the gleam in molecular biologists' collective eye as to where the science is going? I think understanding the molecular basis of develop ment is the big challenge in biology. At present, there is no general theory that can be formulated to explain development; for example, the progress from a fertil ized cell to an adult organism. There is nobody naive enough to believe that there will be a single explana tion for all types of development. Most likely, each system will have some unique features that set it apart, but the basic reactions of DNA to RNA to protein will be a common feature of all systems. Proba bly we will have to work our way through a lot of these systems before a consistent pattern emerges. Another aspect of development that intrigues many people is trying to translate cell phenomenology or organ morphology into molecular terms. For instance, what governs the liver's growth and shape? What regulates the growth of cells? What triggers a cell into the division cycle? All of these questions require mo lecular explanations. Ultimately, they all will be de fined in molecular and chemical terms. Although most molecular biologists think about the 62
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nervous system only in their spare moments, they must wonder about the molecular bases of nervous system development. How are all the right connections made? How is information stored and transmitted among nerve cells? Although difficult, a few labs are using molecular cloning to isolate genes important for the development or functioning of simple nervous systems. Recombinant DNA techniques have moved well beyond the domain of molecular biologists. Recombinant DNA techniques have enabled chemists to alter proteins in ways that could not be done before. This enables them to determine how the changes affect catalysis, folding, substrate binding, ligand binding, recognition by antibody, and so on. I have no doubt that recombinant DNA techniques will cre ate an explosive turning point in chemical studies of protein structure and function. The transition from basic research to applied re search has been very rapid for recombinant DNA techniques thus far. Do you see that same kind of rapid transition for the kind of basic research we have been talking about? I think the impact of basic discoveries to applied research will continue to be rapid. Studies of the immune system provide a good example. Basic scien tists view immune systems as elegant models for the study of development. How does an organism devel op an immune response? What are all the components that are necessary to prevent an organism's cells from making antibodies against its own tissues and yet enable it to mount an immune response against an infinite variety of foreign chemical substances? You can see if you had answers to these questions, you could rapidly translate that understanding into ther apies that intervene or modify the immune response. Allergy is a good example. It seems that an allergic response depends upon the overproduction of a partic ular immunoglobulin. Normally, such immunoglobulins are made in small quantities and directed only against a few substances. But an individual who is allergic to pollen produces that rare immunoglobulin directed against the pollen antigen, and when the two com bine the allergic response is produced. If we knew how to intervene in the production of that abnormal immunoglobulin, or could regulate the T-cells that promote the production of the abnormal immunoglobulin by B-cells, it might be possible to prevent the allergic response at several stages. Many drug companies are actively working in this field. They recognize that if they understood the molecular basis of immunoglobulin gene regulation and how the gene products interact with the various cell types of the immune system, they could try to tailor-make drugs or analogs to alter immune functions. That is clearly one area in which the basic information might be translated into therapeutic action very rapidly. Another example is in the area of hemoglobin diseases. If we knew how to switch on the gene en coding fetal β-globin, we might cure sickle-cell anemia. That might be possible for other diseases as well.
what some people say today, we came out of the controversy in a stronger position to guide the research than if we had ignored or stonewalled it. I have no doubt that had we gone ahead without raising the issues ourselves, strict regulations would have been imposed on us and we would have had no say at all about their content or administration. Do you think anything has changed in terms of the public's becoming any more educated about recombinant DNA except for the perception that there are going to be beneficial products coming from the genetic engineering industry? In the final analysis, I think it was the commercial promise that permitted cloning to move ahead without pressures for strict regulation. Had the benefits to basic research been the only justification, recombinant DNA techniques might have been banned. However, the hype about all the commercial and medical benefits certainly created pressures to allow the work to proceed without too stringent regulations.
A rock-solid faith that recombinant DNA and molecular genetics are leading in directions and to products that cannot now be foreseen On the basic research side? Absolutely. So the pace is going to continue to be rapid? Yes. The therapeutic products that are appearing now are the easy ones. They are the ones that were identified as the likely benefits from the beginning: growth hormone, insulin, interferon, blood clotting factors, vaccines, diagnostics, etc. It is a bit more difficult to identify the second-, third-, and fourth-generation advances, and probably won't be possible until some of the basic problems I talked about are solved. However, there is amongst most scientists an absolute, rock-solid faith that recombinant DNA and molecular genetics are leading in directions and to products that cannot now be foreseen. For that reason most companies believe that they must be involved in the basic work now and that they cannot wait until identifiable product applications are obvious to all. Some researchers seem to have some doubts now about the wisdom of scientists publicly scrutinizing the potential risks of recombinant DNA research in the mid-1970s. The point they make is that the process has implanted a fear of genetic tampering in the public consciousness. Do you have any regrets about how things were handled in those early years? I have no regrets about having raised the issues. I thought it was the right thing to do, and at the time all who raised the question thought it was the right thing to do. That's not to say that I do not have regrets about some of the things we did and about the outcome. What I regret most is that after all the turmoil, no mechanism or procedure survives that could deal with similar issues should they arise again. We might have to go through the same nonsense all over again. I'd like to think that scientists as a group were seen as being responsible in doing what we did. In spite of
The original controvery never made it into the courts, and potential legislation was successfully deflected. That will not be the case concerning deliberate release experiments. Do you worry about legislation or legal actions hindering recombinant DNA research either at the basic level or at the more applied level? Do you see things coming up again that you thought had been put to rest in the 1970s? I don't think the debate will focus on the same issues again. However, I suspect that the deliberate release of organisms into the environment or the use of recombinant DNAs for human gene therapy still will arouse public interest and that will have to be reckoned with. I think the public is still confused and unprepared to evaluate the risks and lack of risks in such activities. It does seem that [author and social critic] Jeremy Rifkin strikes a real chord when he lumps molecular biologists with the nuclear physicists who gave us nuclear weapons and nuclear waste and with the chemists who gave us toxic waste dumps. When he says that molecular biologists are just the next group in a long line of scientists who are going to cause a disaster, people respond to that. Sure they do. But I don't think that argument alone would have gotten him very far. I believe Rifkin has found the note that preys on people's fears that basic genetic research is opening the door to genetic manipulation of humans. He has tried to create the impression that scientists and physicians are on the verge of attempting to modify human germ-line genes. I think scientists in this field know better than Rifkin what the problems and implications of such activities would mean. Nevertheless, he has certainly been successful in implanting his pseudo-scientific charges into the minds of influential people, and that will have to be reckoned with as time progresses. D August 13, 1984 C&EN
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