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pect of constructing bacterial cells, which can be grown easily and inexpen- sively, that will synthesize a variety of biologically produced substances such as antibiotics and hormones; or enzymes that can convert sunlight directly into food substances or usable energy. Per- haps it even provides an experimental basis for introducing new genetic infor- mation into plant or animal cells. It has been clear from the beginning of experimentation in molecular cloning that the construction off some kinds of novel gene combinations may have a po- tential for biological hazard, and the sci- entific community has moved quickly to make certain that research in genetic manipulation would not endanger the public. For a time after our initial experi- ments the pSC101 plasmid was the only vehicle known to be suitable for cloning foreign DNA in E. coli, and our col- leagues asked for supplies with which to pursue studies we knew were of major scientific and medical importance. In- vestigators normally facilitate the free exchange of bacteria and other experi- mental strains they have isolated or de- veloped, but Chang and I were con- cerned that manipulation of certain genes.could give rise to novel organisms whose infectious properties and ecologi- eal effects could not be predicted. In agreeing to provide the plasmid we therefore asked for assurance that our colleagues would neither introduce tu- mor viruses into bacteria nor create anti- biotic-resistance combinations that were not already present in nature; we also asked the recipients not to send! the plas- mid on to other laboratories, so that we could keep track of its distribution. When still other cloning vehicles were CLEAVED TOAD RIBOSOMAL DNA discovered, it became apparent that a more general mechanism for ensuring ex- perimental safety in gene-manipulation research was advisable. The groundwork for such control had been established earlier: the National Academy of Sci- ences had been urged to consider the "possibility that potentially biohazardous consequences might result from wide- spread or injudicious use" of these tech, niques and had asked Paul Berg to form an advisory committee that would con- sider the issue. Berg too had been con- cerned about the potential, hazards of certain kinds of experimentation for some years, and had himself decided to abandon plans to try to introduce genes from the tumor virus SV40 into bacteria • because of the possible danger if the ex- periment were successful. Berg brought together a number of i4- vestigators, including some who were then directly involved in molecular clon- ing, in the spring of 1974. In a report released in July and in a letter to leading professional jpurnals the members of the committee expressed their "concern about the possible unfortunate conse- quences of indiscriminate application" of the techniques and formally asked all, investigators to join themi in voluntarily deferring two types of experiments (which had, as a matter of fact, been avoided by informal consensus up until that time). Experiments of Type I in- volved the construction of novel orga- nisms containing combinations of toxin- producing capabilities or of antibiotic- resistance genes not found in nature. Type 2 experiments involved the intro- duction of DNA from tumor viruses or other animal viruses into bacteria; the committee noted'~ that "such recombinant molecules might be more easily dissemi- CD 42 CLEAVED PLASMID CHIMERAS nated to bacterial populations in humans and other species, and might thus in- crease the incidence of cancer or other diseases." The Academy committee was con- cerned largely because of our inability to assess the hazards of certain experi- ments accurately before the experiments were undertaken. Guidelines for safety had long been available in other areas of potentially hazardous research, such as studies invol+ving known disease-causing bacteria and viruses, radioactive isotopes or toxic chemicals. Because of the new- ness of the microbial gene-manipulation methods, no such guidelines had yet been developed for work in this area, however; there was the possibility that potentially hazardous experiments might proceed before appropriate guidelines could be considered and implemented. We recognized that most work with the new methods did not and would not in- volve experiments of a hazardous nature but we recommended the deferral of ' Type I and Type II experiments until the hazards were more carefully assessed, until it was determined whether or not the work could be undertaken safely and until adequate safety precautions were available. The committee also proposed that an international meeting be held early in 1975 to consider the matter more fully: Such a meeting was held in February at the Asilomar Conference Center near Pacific Grove, Calif. It brought together 86 American biologists and 53 investiga- tors from 16 other countries, who spent three and a half days reviewing progress in the field of molecular cloning and for- mulating guidelines that would allow most types of new hereditary character- istics to be introduced into bacteria and CD30 CD35 CD4 CD 18 -4.2 -3.9 4 •/ GEL ELECTROPHORESIS demonstrates the presence of toad DNA in chimeric plasmids. Fragments of DNA migrate through a gel at different rates under the influence of an electric current, de, pending on their size. Linear molecules of plasmid DNA (right) and the cleavage products of toad ribosomal DNA (Te/t) therefore 10 3.0 have characteristic sizes and migrate characteristic distances in a given time. The bands of DNA, visualized by a fluorescent dye, are photographed in ultraviolet. All five chimeric plasmids (center) contain a plasmid DNA molecule; in addition each chimera in- cludes one or more fragments characteristic of original toad DNA. CLEAVED pSClot PLASMID c

~_ viruses safely: Invited nonscientists from the fields of law and ethics participated in the discussions and decisions at Asilo- mar, along with representatives of agen- cies that provide Federal funds for sci- entific research; the meetings were open to the press and were fully reported. The issues were complex and there were wide differences of opinion on many of them, but there was consensus on three major points. First, the newly develope& clon- ing methods offer the prospect of deal- ing with a wide variety of important sci- entific and medical problems as well as other problems that trouble society, such as environmental pollution and food and energy shortages. Second, the accidental dissemination of certain novel biological combinations may present varying, de- grees of potential risk. The construction of such combinations should proceed only under a graded series of precau- tions, principally biological and physical barriers, adequate to prevent the escape of any hazardous organisms; the extent of the actual risk shoul& be explored'by experiments conducted under strict con- tainment conditions. Third, some experi- ments are potentially too hazardous to be carried out for the present, even with the most careful containment. Future re- search and experience may show that many of the potential hazards considered at the meeting are less serious and less probable than we now suspect. Never- theless, it was agreed that~ standards of protection should be high~ at the begin- ning and that they can be modified later if the assessment of risk changes. Physical containment barriers have long been used in the U.S. space-explo- ration program to minimize the possibil- ity of contamination of the earth by ex- traterrestrial microbes. Containment pro- cedures are also employed routinely to protect laboratory workers and the pub- lic from hazards associated with radio- active isotopes and toxic chemicals and in work with disease-causing bacteria and viruses. The Asilomar meeting for- mulated the additional concept of bio- logical barriers, which involve fastidious . cloning vehicles that are able to propa- c gate only in specialized hosts and equal- ly fastidious bacterial, strains that'~ are unable to live except under stringent laboratory conditions. In the past the scientific community has commonly policed its own actions in- formally, responding to ethical concerns with self-imposed restraint. Usually, but, not always;,society at large has also con- sidered the public well-being in deter- mining how knowledge: obtained by basic scientific research should be ap- plied. Extensive public scrutiny and TOAD DNA . HETERODUPLEX ANALYSIS identifies regions of a toad DNA (black) that have been in- corporated in a chimeric plasmid DNA molecule. DNA isolated from toad eggs and the DNA of the chimera are denatured„that is, each natural double-strand molecule is split into two single strands of DNA, by alkali treatment. The toad and the cbimeric DNA's are mixed together, and any complementary sequences are allowed to find each other. The toad DNA incorporated in the chimeras has nucleotide sequences that are complementary to sequences in the DNA taken directly from the animal source. Those homologous sequences anneal to form heteroduplex double-strand DNA..t6at can be identified in electron micrograpbs. open discussion by scientists andd non- lic discussions initiated by scientists scientists of the possible risks and bene- working in genetic manipulation will be. fits of a particular line of basic research One can hope that the forthright ap- has been rare, however, when (as in this proach and the rigorous standards that case) the hazards in question are only have been adopted! for research in the potential and; for some experiments, cloning of recombinant DNA molecules even hypothetical. As this article is be. will promote a sharper focus on other ing written it is still'too early to know issues relevant to public and environ- what the long-range outcome of the pub- mental safety. PRESENCE OF'TOAD DNA in two separate chimeric plasmid molecules is demonstrated by an electron micrograph made by John F. Morrow at the Stanford University School of Medicine. As is indicated in the drawing (bottom), there are DNA strands from two plas- mids and a strand of toad DNA. The micrograph shows thickened regions of DNA where nucleotide sequences are homologous and two single strands have been annealed. The toad DNA in the chimeras cddes for ribosomes, and the space between the two betero- duplex regions is compatible with the spacing of multiple ribosomal genes in toad DNA. II PLASMID DNA1ll1CKING

The Author STANLEY N. COHEN is associate professor of medicine at the Stanford University School of Medicine. A gradu- ate of Rutgers University and the Uni- versity of Pennsylvania School of Medicine, he joined the Stanford faculty in 1968 after spending several years teaching and doing research in molecular biology at the Albert Einstein College of Medicine. His research has also involved a stint at'the National Institute of Arthri- tis and Metabolic Diseases. A specialist in bacterial plasmids, Cohen was a mem- ber of the National Academy of Sciences committee that recently callod' for the voluntary deferral of' certain potentially hazardous experiments involving recom- binant DNA molecules. Bibliography CONSTRUCTION' OF BIOLOGICALLY FUNCTIONAL BACTERIAL PLASMIDS IN' VITRO. Stanley N. Cohen„Annie C. Y. Chang, Herbert W. Boyer and'Robert B. Helling in Proceedings of the Na- tional Academy o f Sciences o f the United States of America, Vol. 70, No. 11, pages 3240-3244; November, 1973. REPLICATION AND TRANSCRIPTION OF EUxARYoTIc DNA IN ESCHERICHIA COLI. John F. Morrow, Stanley N. Cohen, Annie C. Y. Chang, Herbert W. Boyer, Howard M. Goodman and Robert B. Helling in Proceedings of the National Academy o f Sciences o f the United States of America, Vol. 71, No. 5, pages 1743-1747; May, 1974. POTENTIAL BIOHAZARDS OF RECOMBI~ NANT DNA MOLECULES. P. Berg et al. in Proceedings of the National Acad- emy of Sciences of the United States of America, Vol. 71, No. 7, pages 2595-2599; July, 1974. FIRST ASM CONFERENCE ON E%TRA- CIiROMOSOMAL ELEMENTS IN BACTE- RIn in Microbiology-1974. American. Society for Microbiology, 1975. REPORT OF TIIE WORKING' PARTY ON THE EXPERIMENTAL MANIPULATION OF MICROORGANISMS. Her Majesty's Stationery Office, London;1975.