APPENDIX F CLONED EUKRRY OTIC, DNA Introduction We envisage three ways that recombinant eukaryotic DNA mole- cules could be hazardous. 1. 2. 3. A gene could function in the bacteria in which it is cloned and A DNA component could in someway enhance the virulence A DNA component could infect some plant or arrimal, integrate produce a toxic product. or change the ecological range of the bacterium, in which it is cloned, into its genome, or replicate, or by its expression could produce a modification of the cells of the brganism. The vector with which the DNA is recombined, the source of the eukaryotic DNA, and the kind of experiment used to produce recom- binant molecules are important variables which we have considered. Mainly we discuss potential hazards of laboratory experiments, and these may increase when recombinant DNAs of known function are used for some applied medical, or agricultural purpose. We have commented on this at the end. Our assessment of the biohazards of recombinant DNAs has been made in terms of the class of protection which might be required for any particular experiment, using the different levels defined by the plasmid group. 1. Vectors. The great majority if not all of the in vitro recombinant DNA -_I_ molecules that have been constructed to date have been propagated in E. coli and have, perforce, required plasmids or bacteriophage as their vectors. We have asses sed potential biohazards for various types of donor fragments used with these systems, which we would regard as exemplifying prokaryotic vector and host systems. are illustrated in Table 1. These Many of the future developments in the cloning, amplification, and utilization of eukaryotic DNA sequences will, however, require other vectors and host cells. therapy, for example, can only be achieved by transfer of new se- quences to animal cells, for which the obvious vectors are animal viruses. Similar systems are attractive for furthering the basic molecular biology of eukaryotic cells, both animal and plant, but they immediately present an additional range of potential biohazards. The long term objectives of gene Clearly the discovery or even construction of a DNA vector for simple eukaryotes would be of very great advantage for this work. - 2- Will the hazards attending the use of eukaryotic vectors and host Only cells be greater or less than those arising in experiments where the same DNA fragments are manipulated in prokaryotic systems? time and experience can provide the necessary information on this, but since the transfer of new sequences to animal host cells via ani- mal viruses places the new sequences directly in the type of target area that we wish to avoid when using prokaryotic systems to amplify selected DNA fragments, we can only assume that a potential bio- hazard inherent in a given DNA fragment is likely to be greater when propagated in the animal system. rating which is in general assigned in Table 1 to a given type of DNA fragment in such an instance. Norrr-ally, the conditions under which such experiments might be conducted would at least match those used for work with the animal virus concerned. This is reflected in the higher Plant virus and host cells present some difficulty and we have attempted to make a conservative assessment in the light of corrvllon handling practice for plant viruses. Hence, the consensus was that the systems are less likely to present the degree of biohazard po- tential encountered with animal cell systems, but that it would be prudent to equate them with at least the level accorded to correspond- ing experiments with prokaryotic vectors and host cells. 2. Eukaryotic DNA molecules In general the level of precautions employed in experiments involving incorporation of eukaryotic virus genomes should not be less than that appropriate for the donor virus itself. Its ho t gun" e-xpe riment s involve the pro duc ti on of recombinants be - tween a vector and total eukaryotic DNA. will be used extensively to isolate a wide variety of eukaryotic DNA components. Mammalian DNAs, primates in particular, were rated high in biohazard because DNAs from these sources are more likely to contain infectious agents pathogenic for humans. Shotgun experi- ments involve DNA of unknown function, and introduce into the bac- terium new sequences with unpredictable consequences. These con- siderations place all shotgun experiments in Class 3 or higher re- gardless of the source of DNA. This kind of experimentation Purified DNA components are distinguished in biohazard severity by whether or not they are expressed in the bacterium. assignments may seem high but this conservative assignment reflects our ignorance of how eukaryotic DNAs will function within bacteria. We suggest that a eukaryotic gene for a pro- tein usually will not be transcribed nor translated faithfully inside bacteria without additional genetic manipulations . Some class We have also listed the kinds of conditions and information which would lead to a reclassification of the recombinant DNA. Finally, we describe sorne contract experiments which should be performed to inforin us about the hazard potential of these DNAs. TABLE I CLASS ASSESSMENT FOR DIFFERENT KINDS OF EXPERIlvIENTS DNA 1. Shotgun experiments - DNA or transcripts of RNA DNA: Primate Mammalian 0 the r vertebrate Invertebrate Higher plant Simple eukaryote 2. High risk animal viruses Moderate to low risk animal viruses Plant viruses Purified DNA or transcripts of purified RNA from viruses1 3. Purified eukaryotic sequences - DNA or transcripts of RNA (a) Expressed in host of vector 2 1. Known toxic products 2. Other products (b) Not expressed VECTOR I EUKARYOTIC PROKARY OTI( iNIMAL PLANT 5 4 4 4 4 4 4 3 3 4 3 3 4 3 3 4 3 3 5 5 5 4 4 4 4 3 3 or 4 5 5 5 4 3 or 4 3 4 3 2 Use of partial genomes might lead to reevaluation, 1 The experiments would always have to be done on the initial assump- tion of expre s sion. 2 - 4- The table shows the consensus of the committee on the class of protection required for different types of experiments. was good. group, wide, leaving much to the discretion of the investigator. bers assigned certain experiments to class 6, but we later agreed that this would be hard to implement; nevertheless we all felt that there were sorne experiments, such as joining a high risk virus to an animal vector, which would require very strong scientific justifi- cation to be done at all. Concordance The levels of protection are those defined by the plasmid It was recognized that the definition of class 3 protection is Some mim- Purified DNA derived from any of these experiments has not been considered separately. of cloned recombinant DNA molecules it would be prudent to destroy such preparations before disposal. In view o€ our ignorance of the infectivity Reassessment of Hazard Class Assignments The initial classification of experiments will be made largely in ignorance of actual hazards and should be reevaluated in the light of subsequent experience. Some examples of instances in which these classifications could be reassessed are: A. Possible reassignment to a lower class. 1. 2. 3. "Safe" bacterial carriers or plasmid vectors are used; ex- amples are given in the plasmid group's report. It is demonstrated that the eukaryotic DNA segment is not ex- pres sed. Individual recombinant DNAs from a shotgun experiment are shown to contain only genes of known function and these are expected to be minimally hazardous. terialized (e. g. , recombinant plasmid DNA containing human DNA frag- ments is shown to be incapable of transforming or infecting human cells). Other examples are given in the plasmid group's report. plasmid contains only genes which are expected to present lesser ha- zards than the whole viral genome. 4. Tests demonstrate that anticipated biohazards have not ma- 5. The portion of an animal virus DNA in a particular recombinant B. Possible reassignment to a higher class. 1. 2. For special purposes, more pathogenic bacteria or less de- It is discovered that the ecological potential or virulence of sirable vectors (e. g. , F factors) are used. the bacterial carrier has been increased by the presence of the re- combinant pla smid. - 5- 3. Alterations of the plasmid or the carrier are initiated or accomplished which would increase the probability of expr-es sion of the eukaryotic DNA. A recombinant plasmid derived from a shotgun exTeriment is found to contain seqgences homologous to eukaryotic virus genomes. A strain co2sidered to he relatively safe is mutagenizsd. 4. 5. - Hazards Associated with Large Scale Applications Although it may be that eukaryotic genes are not normally trans- scribed and/or translated with fidelity in bacteria vve pre SLIC that such conditions can be obtained by genetic manipulation of the bacterial genome, or of the vector-eukaryote hybrid DNA. Numerous applications of this kind of eukaryotic gene activity have been suggested - for example, the bacterial production of insulin in pharmaceutical factories. the growth of very large numbers of the relevant bacteria, and the eukaryotic gene products they contain may well be hazardous to the general population. These applications will generally involve The problems of containment associated with these applications We therefore set them apart from the hazard ratings given are likely to be increased substantially over those considered pre- viously. above. We recommend that such applications be undertaken only after it can be demonstrated that the bacteria are "safe"; that is, they will not be hazardous even ii they escape the confines of their in- tended use. by the plasmid group. The concept of safe bacteria is discussed in the report - 6- APPENDIX Sonie Experiments Relevant to Hazard Assessment 1. Will a eukaryotic gene function properly in a bacterium? From the available data, it seems unlikely that E. coli RNA poly- merase will generally recognize eukaryotic proniotor, initiation or termination sequences in DNA, nor do E. coli translation factors generally recognize starting signals in eukaryotic mRNAs. This is certain to be an area of intense investigation. 2. answered with viral nucleic acids since a single infective event is greatly amplified and readily assayed. acid should be tested. The survival of the biological potency of DNA may be assayed in higher organisms by using transformable bacteria growing in the animal or plant. Can DNA and RNA infect animals or plants? This could best be All means of entry of the nucleic 3. from bacterial cells to animals or plants? sensitive probe would be a hybrid between a plasmid and a plant or animal viral genome. Knowledge of the answer to question 2 is essential since transmission could occur by the release of DNA or RNA from bacterial cells. teria containing the hybrid molecule should be tested. Can hybrid DNA molecules or their transcripts be transmitted Again probably the most All forms of transmission from the bac- 4. or plant cell? tected form of the DNA, may be more readily taken up by eukaryotic cells. between bacteriophage X and a plant and animal viral genome. Can a hybrid DNA molecule in a phage particle infect an animal A phage particle, being a highly condensed and pro- Again probably the most sensitive probe would be a hybrid 5. Can "safe1'vectors or cloning cells be constructed? A I1safe" vector or cloning cell may be designed to self destruct upon ''escaping'' from a laboratory. tations in essential functions may be incorporated into the cloning cell and vector. gene without the methylase gene into the vector. only then propagate in the corresponding methylating cell. For example a number of conditional lethal mu- Also one might incorporate a restriction endonuclease Such a vector could 6. ments, or any other DNA? Can human cells in culture be transformed by human DNA frag- 7. have altered ability to colonize any eukaryote? Will bacteria containing eukaryotic DNA recombinant molecules, - 7- 8. ments be transfered to other bacterial cells? How frequently carL the plasmids or phage used in cloning experi- Sydney Brenner Donald D. Brown Robert H. Burris Dana Carroll Ronald W. Davis David S. Hogness Kenneth Murray Raymond C. Valentine ADDENDUM The committee concerned with eukaryotic DNA compiled its report as a working paper based on the concept that this report would be presented to the entire conference for evaluation. carries a table which assigns numbers to classes of experiments. These numbers correspond to the classes of containment and control proposed in thz report from the Plasmid Group. assigned have engendered most controversy regarding the report, therefore we would like to explain them and their intent as follows: The report The specific numbers 1. after substantial discussion and there was remarkable uniformity in the risk assignment by committee members. The numbers were derived by consensus within the committee 2. level of hazard to "shotgun" experiments, because of their inherent unpredictability; because of potential hazards arising from homology with human DNA and because of the hazard that primate material might carry slow inc r e as e s . The general reaction of the cornmittee was to assign a higher and to experiments utilizing DNA from primates, 3. side, because ethical considerations dictated that scientists perform experiments cautiously until further data on inherent hazards were established. The assignments of hazard generally used on the conservative 4. The committee particularly stresses that the numbers assigned are illustrative, that there may be substantial variation in hazard within a category, and that the numbers assigned should in no sense be considered immutable. The document suggests information which would dictate upward or downward reclassification of risk. in information generally would dictate a downward reclassification. An increase 5. revised by that committee. The report is advisory only to the Berg committee and may be 6. a redefinition of terms which would embrace classes 1 and 2 (all numbers still to be interpreted in terms of the control and containment criteria of the dasmid renortl in a new class entitled low hazard. class 3 as The eukaryotic DNA committee would be entirely agreeable to -.I. L intermediate hazard, class 4 as high hazard, and class 5 as special hazard. 7. old or alternative new classification for consideration by the Berg Committee with the provisa that any assignment the Berg Committee makes of specific experiments to hazard classes be reexamined and revised in 1 year and subsequently as needed. The committee suggests that the conference approve either its ADDENDUM Modification of Section 3B3 of Recombinant DNA Report 3. B.3, Eukaryotic DXA - In the low risk category are experiments involving fusion of prokaryotic vectors with DNA from all eukaryotes except warm-blooded vertebrates. Purified DNAs whose functions are judged to be non-toxic may be reconbined under low risk conditions. Moderate risk experiments involve the joining of uncharacterized DNA from warm-blcaded vertebrates to prokaryotic vectors. When these experiments use a vector or bacterium judged to be "safe", they can be reassessed to the low risk category- with a high risk include the fusion of prokaryotic vectors with eukaryotic or prokaryotic genes which are likely to result in the expression of a toxic product by the bacterial host, These experiments can be reassessed to a lowerrisk category by using a safe vector and a safe bacterial strain for cloning. Experiments Donald D. Brown 3/3/75