Medical Research Council committee draws up guidelines for research into recombinant DNA
*Ms Mitchell is a graduate student and Dr. Kaplan is department chairman in the department of biology, University of Ottawa Reprint requests to: Ms Mary Mitchell, Department of biology, University of Ottawa
zards inherent in this field of science. Many people believe it is necessary to take a stand against experiments with recombinant DNA molecules until we are more able to predict their properties; they maintain that, as we are still far from understanding the control mechanism in cells of higher organisms, we can hardly predict the effects of tampering with them. Sinsheimer' has suggested that the effects of breaking the natural biologic barrier between prokaryotic and eukaryotic cells could be disastrous, affecting evolution in unpredictable ways - bacteria could, perhaps, acquire the ability to act as reservoirs for common eukaryotic viruses. A more immediate hazard is the production of new and potentially dangerous organisms that would threaten man and other species if they were inadvertently allowed to escape from the laboratory. Particular risk is attached to the shotgun type of experiment in which the DNA of an organism is segmented and randomly inserted in bacteria. Fragments carrying the DNA of interest could also carry the genes for a virus or toxin. Most cells are thought to contain latent viruses, possibly oncogenic, and unexpressed genes for toxic products. Transfer of these genes from their natural environment might mean that they were no longer subject to repressive controls and this might facilitate their expression. The threat becomes more real when we consider that the most commonly used microorganism is Eseherichia ccli, which colonizes the human gut and throat. The logic behind this is that E. coil has been extensively studied and its genome completely mapped. Its proponents claim this means that hazards could be more accurately estimated. Even if "accidental" recombinants are not produced, one must consider the possible consequences for an individual if, for example, a particular strain of E. coli that was able to produce insulin in excess were to grow in his intestine.
FIG. 1-Cleavage sites for restriction endonuclease EcoRi. Note symmetric sequences A-A-T-T in upper strand of DNA and T-T-A-A- In lower strand. Also to be noted are the two "sticky" tails -A-A-T-T- and -T-T-A-A- in right and left halves of lower part of figure.
was approved in the United Kingdom. In Canada a committee chaired by Professor Louis Siminovitch of the University of Toronto and chief of the department of medical genetics at Toronto's Sick Children's Hospital reported to the Medical Research Council (MRC) on prospective guidelines for the conduct of recombinant DNA research,6 some of which is about to begin or is already in progress. Like its predecessors, the MRC report classifies different types of experiments according to their potential hazard and recommends appropriate physical and biologic containment levels. Experiments are categorized according to the source of the DNA to be recombined or cloned and whether or not it has been purified and genetically characterized. (Actually the only truly "pure" DNA is that which has been chemically synthesized. DNA purified from cells probably contains gene sequences other than those desired.) The MRC guidelines
Need for research guidelines
In 1974 the National Academy of Science in the US called for a voluntary moratorium on the more hazardous types of recombinant DNA research. This was partially lifted the next year after a conference at Asilomar California, at which it was agreed that work on construction of recombinant DNA molecules should proceed, provided adequate biologic and physical barriers were used to contain the newly created organisms.4 A National Institutes of Health committee formulated a set of guidelines for conduct of experiments with recombinant DNA', and a similar set of guidelines
Biologic containment is effected by using disabled organisms with a severely limited capacity to survive outside special laboratory conditions. It is obviously necessary to specify biologic containment levels for a particular system, and the MRC report addresses itself to the widely used E. coli strain K12 vector-host systems. Three levels (low, medium and high) of biologic containment are specified, in terms of various types of modifications of host, vector or both. It is recommended that certain hazardous experiments should be undertaken only with "crippled" host-vector systems that have been certified as such by the MRC.
A particularly disturbing trend is emerging that well illustrates the dilemma of scientific responsibility: certain individuals and their institutions are attempting to patent the crippled strains they have developed. Physical containment refers to physical facilities and safe handling techniques designed to prevent the organisms escaping from the laboratory or infecting laboratory staff. The report designates six levels, A-F. * Level A: No more is required at this level than the use of practices that should be routine in a medical microbiology laboratory - inactivation of waste before disposal, regular disinfection of laboratory work surfaces, good hygienic practice and good laboratory design ensuring an adequate airflow through the laboratory. Experiments using purified prokaryotic DNA that will not contribute to pathogenicity or toward the introduction into the bacterial host cell of a foreign gene that does not occur naturally in the host are permitted under level A containment. The latter restriction may be circumvented if a higher level of biologic containment is used. * Level B: In addition to the safe practices required for level A, all manipulation likely to produce an aerosol must be carried out in an exhaust or vertical laminar airflow hood, with decontamination carried out by autoclaving or incineration of all contaminated material and prohibition of oral pipetting. An emergency plan for dealing with spills and other laboratory accidents should be formulated. Certain experiments involving unpurified DNA are permitted under level B containment provided the DNA be from a
CMA JOURNAL/APRIL 9, 1977/VOL. 116 803
nonpathogenic microorganism, plant virus, plant or invertebrate and a high level of biologic containment be ensured. * Level C: A segregated room is required from which all air is exhausted directly to outside or circulated after passage through a high-efficiency particulate air (HEPA) filter. Only authorized persons should have access to the work area. Permitted are experiments already described with a lower level of biologic containment. Using higher biologic containment, level C physical containment is sufficient for the conduct of experiments with unpurified DNA (but known to be nonpathogenic) from invertebrates, cold blooded vertebrates and plant viruses. Purified DNA from animal viruses or warm-blooded vertebrates may be used under higher biologic containment. * Level D: Airflow must be directed through the laboratory and thence directly to outside after HEPA filtration. Elaborate medical precautions are recommended. Personnel should be issued with cards that would indicate to any physician consulted that they were exposed to hazardous agents. The senior investigator should ensure that any unexplained employee's absence is investigated immediately and he should have access to relevant medical information. Before a person is employed, blood specimens should be collected and stored so that subsequent specimens can be collected as necessary and antibody titres determined. Experiments can be conducted with DNA from warm-blooded invertebrates or certain animal viruses provided high-level biological containment systems are used. * Level E: In addition to the conditions specified for level D, entry to the laboratory must be through a ventilated air lock. * Level F: An isolation unit geographically separated from other areas is necessary with a special containment area maintained at a pressure negative to that of the corridors. The report recommends that certain experiments with a high degree potential hazard should be assessed individually by the MRC (such as experiments with DNA likely to carry a determinant of pathogenicity). It also suggests that three classes of experiment be forbidden at present: * Deliberate creation of DNA expected to make harmful products.
* Deliberate release into the natural environment of an organism containing recombinant DNA. * Use of recombinant DNA to transfer drug resistance to microorganisms not known to acquire it naturally, if such an acquisition could compromise the effective use of the drug in medicine or agriculture.
Erythrociii (erythromycin stearate, Abbott)
Future problems The MRC guidelines are meritorious. The problem arises in enforcing thorn. The report places the main responsibility on the principal investigator to ensure that adequate facilities are available and proper procedures are followed in his laboratory. For further enforcement it is suggested that research institutions could establish biohazards coinniittees. This still assumes, however, responsibility on the part of the scientist. The MRC, of course, intends to review containment procedures used in research performed under MRC operating grants, and the NRC has already indicated that it will follow suit - but how can the guidelines be enforced in, for example, pharmaceutical research laboratories? A law requiring compliance with the NIH guidelines5 has been presented to the US Senate; similar legislation will be required in Europe and Canada. Many biologists advocate either federal action to ensure that adequate containment levels are universally used or that all extremely hazardous research be carried out in one laboratory. This is a controversial issue as it could lead to severe restrictions of scientific freedom. In any case, if Sinsheimer is correct in his belief that the risk is not in the particular gene inserted but in the very fact of putting eukaryotic genes into prokaryotes then all the rules so far constructed mean remarkably little. With the advent of recombinant DNA technology we are seeing the transformation of biology from a primarily analytic to a synthetic science. Nothing can be more far reaching than the creation of new forms of life. As Chargaff7 has said, "You can stop splitting the atom; you can stop visiting the moon; you can stop using aerosols; you may even decide not to kill entire populations by the use of a few bombs. But you cannot recall a new form of life..
500-mg Tablet b d. Convenient q 12 h dosage for bacterial respiratory tract infections Availability: Each Filmtab* tablet contains: erythromycin (as the stearate) 500 mg buffered with sodium citrate. Available in bottles of 50 Filmtab tablets. Dosage: 500 mg every 12 hours is the usual dose. Dosage may be increased up to 4 g or more per day, according to the severity of the infection. In the treatment of streptococcal infections, a therapeutic dosage of erythromycin should be administered for at least 10 days. In continuous prophylaxis of streptococcal infections in persons with a history of rheumatic heart disease, the dose is 250 mg twice a day. Contraindications: Known hypersensitivity to erythromycin. Adverse Effects: The most frequent adverse effects of oral erythromycin preparations are gastrointestinal, such as abdominal cramping and discomfort and are dose-related. Nausea, vomiting, and diarrhea occur infrequently with the usual oral dose. During prolonged or repeated therapy there is a possibility of overgrowth of non-susceptible bacteria or fungi. If such an infection occurs, the drug should be discontinued and appropriate therapy instituted. Mild allergic reactions, such as urticaria and other skin rashes, have occurred. Serious allergic reactions, including anaphylaxis, have been reported.
References 1. MAUGH TH: The artificial gene: it's syn-
thesized and it works in cells. Science 194: 44, 1976 2. MARIANS KJ, wu R, STAwINsII J, Ct al: Cloning of synthetic DNA. Nature 263: 744, 1976 3. Si.ssszsMEa RL: On coupling inquiry and wisdom. Fed Proc 35: 2540, 1976 4. BERG P, BALTIMORE D, BRENNERs 5, et al: Asilomar conference on recombinant DNA
molecules. Science 188: 991, 1975
5. US Department of Health, Education and Welfare, National Institutes of Health: Guidelines br Research Involving Recombinant DNA Molecules, June 23, 1976 6. Report to the Medical Research Council of Canada from its ad hoc committee: Guidelines for Handling Recombinant DNA Molecules and Animal Viruses and Cells, January 1977 7. CHARGAFF E: On the dangers of genetic meddling. Science 192: 938, 1976
804 CMA JOURNAL/APRIL 9, 1977/VOL. 116
RD. TM.
*Ms Mitchell is a graduate student and Dr. Kaplan is department chairman in the department of biology, University of Ottawa Reprint requests to: Ms Mary Mitchell, Department of biology, University of Ottawa
zards inherent in this field of science. Many people believe it is necessary to take a stand against experiments with recombinant DNA molecules until we are more able to predict their properties; they maintain that, as we are still far from understanding the control mechanism in cells of higher organisms, we can hardly predict the effects of tampering with them. Sinsheimer' has suggested that the effects of breaking the natural biologic barrier between prokaryotic and eukaryotic cells could be disastrous, affecting evolution in unpredictable ways - bacteria could, perhaps, acquire the ability to act as reservoirs for common eukaryotic viruses. A more immediate hazard is the production of new and potentially dangerous organisms that would threaten man and other species if they were inadvertently allowed to escape from the laboratory. Particular risk is attached to the shotgun type of experiment in which the DNA of an organism is segmented and randomly inserted in bacteria. Fragments carrying the DNA of interest could also carry the genes for a virus or toxin. Most cells are thought to contain latent viruses, possibly oncogenic, and unexpressed genes for toxic products. Transfer of these genes from their natural environment might mean that they were no longer subject to repressive controls and this might facilitate their expression. The threat becomes more real when we consider that the most commonly used microorganism is Eseherichia ccli, which colonizes the human gut and throat. The logic behind this is that E. coil has been extensively studied and its genome completely mapped. Its proponents claim this means that hazards could be more accurately estimated. Even if "accidental" recombinants are not produced, one must consider the possible consequences for an individual if, for example, a particular strain of E. coli that was able to produce insulin in excess were to grow in his intestine.
FIG. 1-Cleavage sites for restriction endonuclease EcoRi. Note symmetric sequences A-A-T-T in upper strand of DNA and T-T-A-A- In lower strand. Also to be noted are the two "sticky" tails -A-A-T-T- and -T-T-A-A- in right and left halves of lower part of figure.
was approved in the United Kingdom. In Canada a committee chaired by Professor Louis Siminovitch of the University of Toronto and chief of the department of medical genetics at Toronto's Sick Children's Hospital reported to the Medical Research Council (MRC) on prospective guidelines for the conduct of recombinant DNA research,6 some of which is about to begin or is already in progress. Like its predecessors, the MRC report classifies different types of experiments according to their potential hazard and recommends appropriate physical and biologic containment levels. Experiments are categorized according to the source of the DNA to be recombined or cloned and whether or not it has been purified and genetically characterized. (Actually the only truly "pure" DNA is that which has been chemically synthesized. DNA purified from cells probably contains gene sequences other than those desired.) The MRC guidelines
Need for research guidelines
In 1974 the National Academy of Science in the US called for a voluntary moratorium on the more hazardous types of recombinant DNA research. This was partially lifted the next year after a conference at Asilomar California, at which it was agreed that work on construction of recombinant DNA molecules should proceed, provided adequate biologic and physical barriers were used to contain the newly created organisms.4 A National Institutes of Health committee formulated a set of guidelines for conduct of experiments with recombinant DNA', and a similar set of guidelines
Biologic containment is effected by using disabled organisms with a severely limited capacity to survive outside special laboratory conditions. It is obviously necessary to specify biologic containment levels for a particular system, and the MRC report addresses itself to the widely used E. coli strain K12 vector-host systems. Three levels (low, medium and high) of biologic containment are specified, in terms of various types of modifications of host, vector or both. It is recommended that certain hazardous experiments should be undertaken only with "crippled" host-vector systems that have been certified as such by the MRC.
A particularly disturbing trend is emerging that well illustrates the dilemma of scientific responsibility: certain individuals and their institutions are attempting to patent the crippled strains they have developed. Physical containment refers to physical facilities and safe handling techniques designed to prevent the organisms escaping from the laboratory or infecting laboratory staff. The report designates six levels, A-F. * Level A: No more is required at this level than the use of practices that should be routine in a medical microbiology laboratory - inactivation of waste before disposal, regular disinfection of laboratory work surfaces, good hygienic practice and good laboratory design ensuring an adequate airflow through the laboratory. Experiments using purified prokaryotic DNA that will not contribute to pathogenicity or toward the introduction into the bacterial host cell of a foreign gene that does not occur naturally in the host are permitted under level A containment. The latter restriction may be circumvented if a higher level of biologic containment is used. * Level B: In addition to the safe practices required for level A, all manipulation likely to produce an aerosol must be carried out in an exhaust or vertical laminar airflow hood, with decontamination carried out by autoclaving or incineration of all contaminated material and prohibition of oral pipetting. An emergency plan for dealing with spills and other laboratory accidents should be formulated. Certain experiments involving unpurified DNA are permitted under level B containment provided the DNA be from a
CMA JOURNAL/APRIL 9, 1977/VOL. 116 803
nonpathogenic microorganism, plant virus, plant or invertebrate and a high level of biologic containment be ensured. * Level C: A segregated room is required from which all air is exhausted directly to outside or circulated after passage through a high-efficiency particulate air (HEPA) filter. Only authorized persons should have access to the work area. Permitted are experiments already described with a lower level of biologic containment. Using higher biologic containment, level C physical containment is sufficient for the conduct of experiments with unpurified DNA (but known to be nonpathogenic) from invertebrates, cold blooded vertebrates and plant viruses. Purified DNA from animal viruses or warm-blooded vertebrates may be used under higher biologic containment. * Level D: Airflow must be directed through the laboratory and thence directly to outside after HEPA filtration. Elaborate medical precautions are recommended. Personnel should be issued with cards that would indicate to any physician consulted that they were exposed to hazardous agents. The senior investigator should ensure that any unexplained employee's absence is investigated immediately and he should have access to relevant medical information. Before a person is employed, blood specimens should be collected and stored so that subsequent specimens can be collected as necessary and antibody titres determined. Experiments can be conducted with DNA from warm-blooded invertebrates or certain animal viruses provided high-level biological containment systems are used. * Level E: In addition to the conditions specified for level D, entry to the laboratory must be through a ventilated air lock. * Level F: An isolation unit geographically separated from other areas is necessary with a special containment area maintained at a pressure negative to that of the corridors. The report recommends that certain experiments with a high degree potential hazard should be assessed individually by the MRC (such as experiments with DNA likely to carry a determinant of pathogenicity). It also suggests that three classes of experiment be forbidden at present: * Deliberate creation of DNA expected to make harmful products.
* Deliberate release into the natural environment of an organism containing recombinant DNA. * Use of recombinant DNA to transfer drug resistance to microorganisms not known to acquire it naturally, if such an acquisition could compromise the effective use of the drug in medicine or agriculture.
Erythrociii (erythromycin stearate, Abbott)
Future problems The MRC guidelines are meritorious. The problem arises in enforcing thorn. The report places the main responsibility on the principal investigator to ensure that adequate facilities are available and proper procedures are followed in his laboratory. For further enforcement it is suggested that research institutions could establish biohazards coinniittees. This still assumes, however, responsibility on the part of the scientist. The MRC, of course, intends to review containment procedures used in research performed under MRC operating grants, and the NRC has already indicated that it will follow suit - but how can the guidelines be enforced in, for example, pharmaceutical research laboratories? A law requiring compliance with the NIH guidelines5 has been presented to the US Senate; similar legislation will be required in Europe and Canada. Many biologists advocate either federal action to ensure that adequate containment levels are universally used or that all extremely hazardous research be carried out in one laboratory. This is a controversial issue as it could lead to severe restrictions of scientific freedom. In any case, if Sinsheimer is correct in his belief that the risk is not in the particular gene inserted but in the very fact of putting eukaryotic genes into prokaryotes then all the rules so far constructed mean remarkably little. With the advent of recombinant DNA technology we are seeing the transformation of biology from a primarily analytic to a synthetic science. Nothing can be more far reaching than the creation of new forms of life. As Chargaff7 has said, "You can stop splitting the atom; you can stop visiting the moon; you can stop using aerosols; you may even decide not to kill entire populations by the use of a few bombs. But you cannot recall a new form of life..
500-mg Tablet b d. Convenient q 12 h dosage for bacterial respiratory tract infections Availability: Each Filmtab* tablet contains: erythromycin (as the stearate) 500 mg buffered with sodium citrate. Available in bottles of 50 Filmtab tablets. Dosage: 500 mg every 12 hours is the usual dose. Dosage may be increased up to 4 g or more per day, according to the severity of the infection. In the treatment of streptococcal infections, a therapeutic dosage of erythromycin should be administered for at least 10 days. In continuous prophylaxis of streptococcal infections in persons with a history of rheumatic heart disease, the dose is 250 mg twice a day. Contraindications: Known hypersensitivity to erythromycin. Adverse Effects: The most frequent adverse effects of oral erythromycin preparations are gastrointestinal, such as abdominal cramping and discomfort and are dose-related. Nausea, vomiting, and diarrhea occur infrequently with the usual oral dose. During prolonged or repeated therapy there is a possibility of overgrowth of non-susceptible bacteria or fungi. If such an infection occurs, the drug should be discontinued and appropriate therapy instituted. Mild allergic reactions, such as urticaria and other skin rashes, have occurred. Serious allergic reactions, including anaphylaxis, have been reported.
References 1. MAUGH TH: The artificial gene: it's syn-
thesized and it works in cells. Science 194: 44, 1976 2. MARIANS KJ, wu R, STAwINsII J, Ct al: Cloning of synthetic DNA. Nature 263: 744, 1976 3. Si.ssszsMEa RL: On coupling inquiry and wisdom. Fed Proc 35: 2540, 1976 4. BERG P, BALTIMORE D, BRENNERs 5, et al: Asilomar conference on recombinant DNA
molecules. Science 188: 991, 1975
5. US Department of Health, Education and Welfare, National Institutes of Health: Guidelines br Research Involving Recombinant DNA Molecules, June 23, 1976 6. Report to the Medical Research Council of Canada from its ad hoc committee: Guidelines for Handling Recombinant DNA Molecules and Animal Viruses and Cells, January 1977 7. CHARGAFF E: On the dangers of genetic meddling. Science 192: 938, 1976
804 CMA JOURNAL/APRIL 9, 1977/VOL. 116
RD. TM.
