plasmid o r A and i t s int roduct ion i n t o -E. -c o l i . Const ruct ion of hybr ids betweenE. c o l i phage or plasmid DNA, and unknown genes from Y. pestis, B. anthracis,or B. abortus, when the hybrid is introduced into E. c o l i . See Appendix B foradditional examples.6. Class V I Experiment: Class V I includes experiments i n which the biohazardsare judged to be of such great potential severity as to preclude performance ofthe experiment a t the present time under any circumstances, and regardless ofcontainment conditions.Example of Class V I Experiment: The introduction by any means of the genesf o r botulinum toxin biosynthesis i n t o -E. -c o l i . See Appendix B f o r addi t iona lexamples.C. Summary of ClassificationWe have categorized experiments involving the construction of geneticallyaltered microorganisms into s i x classes. The assignment of experiments toClasses I and I1 involves l i t t l e d i f f i c u l t y , since genetic exchange between theorganisms used occurs normally. Classes 111, I V and V experiments, however,primarily include the construction of genetically altered microorganisms whichuse donor and recipients that ordinarily do not exchange genetic material. Werecognize that i n many specific instances the distinction between a Class I11and a Class I V or between a Class I V and Class V experiment w i l l be d i f f i c u l t tomake since these classes include experiments i n which the potential biohazardscannot be t o t a l l y assessed. Ultimately, the distinction w i l l depend upon theecology of the recipient bacterial species, the nature of the cloning vehicleand the likelihood of phenotypic changes resulting from introduction of there combinant DNA.A natural tendency is to consider changes i n pathogenicity as the primarybiohazard concern since these come to mind most readily when considering microorganisms;other changes which may affect the fundamental ecological potential,adaptability, metabolism, etc. of a recipient organism may be more subtle andmuch more d i f f i c u l t to assess than pathogenicity. However, these alterationsmay potentially present an equal or greater biohazard. We can offer only ar e l a t i v e l y few guidelines to help an investigator i n determining the classassignment of an experiment i n Classes 111, I V or V; perhaps the most c r i t i c a lis the extent of characterization of the genetic material being employed i n theexperiment since we believe that the potential biohazards of a purified and wellcharacterizeddonor DNA species are more easily assessed than the biohazardsinherent i n the introduction of a random assortment of DNA fragments.-111. -CONTAINMENT PRINCIPLES AND PROCEDUNSA. Introduction and General RecommendationsBiological safety and environmental control programs for dealing withpathogenic bacteria have been implemented i n c l i n i c a l and biomedical researchlaboratories for many years ( r e f s . 1-12). Once a potential biohazard has beendefined and the r i s k has been assessed, the major t h r u s t of the proceduresemployed to minimize the biohazard involves steps to l i m i t r i s k to the laboratoryworker and to prevent the escape of potentially hazardous biologicalmaterial.Many of the basic problems of containment t h a t face an investigator studyingrecombinant DNA i n a microbial species are similar to those faced i n every medicalmicrobiology laboratory. A c l i n i c a l specimen received for microbiological analysismay contain an etiologic agent ranging from those of ordinary potential hazardto those which may require the most stringent conditions for t h e i r containment.One cannot be certain u n t i l the etiologic agent is isolated and its known pathogenicity(i.e. its potential hazard) assessed. By the same token, an investigatorwho employs a random assortment of DNA molecules for construction of recombinantDNA molecules could, a t l e a s t i n theory, i s o l a t e a variety of novel transformantbacterial clones which range i n t h e i r potential biohazard. The following safetyconsiderations are applicable to a l l procedures involving etiological agents i nthe c l i n i c a l laboratory. As such they may be considered as prudent standard proceduresfor those working with bacteria containing recombinant DNA molecules.Obviously, those investigators working with animal or plant viruses w i l l need tos a t i s f y the special containment problems inherent i n the laboratory manipulationof these agents.The procedures l i s t e d below are a r e i t e r a t i o n of long-standing microbiologicalpractices and simply reenforce the concept that microbiological safety is amatter of good working habits. A l l of the general recommendations l i s t e d beloware desirable for all classes of experiments, although we recognize that they a r enot specifically needed for the safe handling or containment of all agents.1. Consequently, our primary recommendation for containment of potential biohazardsis that a l l individuals planning research with recombinant DNA moleculesi n bacteria receive adequate training i n microbiology. Such training should notbe construed to mean t h a t one needs to learn only aseptic techniques or the proceduresfor handling potentially infectious material. Rather, investigatorscannot afford to ignore the basic biology of the microorganism -- its ecology,innate pathogenicity, physiology, growth requirements, etc. In short, an investigatormust t r y to think i n microbiological terms before i n i t i a t i n g experimentst h a t could potentially a f f e c t the basic ecology and/or pathogenic potential of anorganism that serves as a c a r r i e r for a recombinant DNA molecule. The microorganismis not simply a "warm body" to house a recombinant DNA molecule of i n t e r e s t .It is axiomatic that no safety f a c i l i t i e s or equipment (no matter now sophisticated)can take the place of an investigator's responsibility. In terms ofbiological safety, the principal investigator eannot delegate, reassign, abandonor ignore his or her responsibility t h a t adequate safety training be given to a l llaboratory personnel. We have appended a list of books and other publicationswhich deal with the general topics of laboratory safety, biohazards i n biologicalresearch and the handling of specific bacterial agents which may prove useful asa source of specific information.2. As a general principle, doors to laboratories i n which potentially biohazardousmaterial is handled should be kept closed.3. Eating, drinking or smoking in the laboratory is undesirable and i n areas i nwhich potentially biohazardous material is handled should be specifically forbidden.4. The most frequent causes of laboratory acquired infections are accidentaloral aspiration of infectious material through a pipette, accidental inoculationwith syringe needles and animal b i t e s (10,ll). A further important causeof both laboratory acquired infections and contamination of the environment isaerosols from centrifugation, blending, loose needles on syringes and even theimproper flame s t e r i l i z a t i o n of contaminated inoculating loops and needles.(see chapter s by Dimmick -et .-al . , r e f . 1) A s minimal recommendations, handwashingby laboratory personnel should be encouraged and direct mouth pipetting should be -discouraged. The use of cotton plugged pipettes may be acceptable for agents oflow or moderate hazard but a mechanical pipetting device is preferable. Specialaerosol precautions are generally not required for most bacterial species, butt h e i r use deserves careful consideration.5. Bacterial cultures and potentially hazardous DNA should be disinfected ors t e r i l i z e d by autoclaving. The laboratory should be cleaned, work surfaces decontaminatedand a l l contaminated material placed i n discard pans (preferablycovered) containing a suitable disinfectant or autoclaved a t the end of the day.The use of specific disinfectants cannot be recommended here, since they w i l l varyfrom bacterial species to bacterial species and, additionalJy, must be capable ofrendering nucleic acid solutions "non-infectious". One should not accept manufactu r e r ' s claims for disinfectant effectiveness -- there is no substitute for a usetestevaluation performed against the microorganism and nucleic acid solutions processedi n the laboratory.6 . Any research group working with agents with a known or potential biohazardshould have an emergency plan, including a clean-up procedure to follow i f anaccident contaminates personnel or environment. Here again, the principal investi g a t o r must insure that everyone i n the laboratory is familiar with both thepotential hazards of the work and the emergency plan.7. If a research group is working with a known bacterial pathogen for which avaccine is available, a l l workers should be vaccinated. Immunization is not, however,a license for procedural short-cutsnor a substitute for safe laboratorypractice .B. Levels of ContainmentThe containment procedures proposed are designed to match the previouslydefined classes of experiments involving novel recombinant bacteria.Since containment cannot be absolute, the rationale underlying these containmentrecommendations is that the greater the potential biohazard, the more s t r i n g e n tshould be the containment. In our judgment, each level of containment implies anacceptable level of protection for laboratory workers and an acceptably low probabi l i t y of escape for the organisms involved.Class I Experiments: Requires no special containment other than practice of standardaseptic technique (i.e. use of procedures to maintain pure cultures and disinfectionof discarded materials).Class I1 Experiments: The basic c r i t e r i a for t h i s category are those minimaloperating procedures employed i n a c l i n i c a l microbiology laboratory. These are:1. Eating, drinking and smoking are forbidden i n the laboratory.2 . Laboratory coats are required during handling of biohazardousmaterial. These should not be worn outside the work area.--Cotton-plugged pipettes or mechanical pipetting devices arerequired. The l a t t e r are preferable.Routine disinfection of work surfaces and prompt disinfectionor s t e r i l i z a t i o n of a l l contaminated material should be carriedout.Immunization of personnel i s requi red f o r experimenting- wi th-S. typhi, V. cholerae, C. diphtheriae and C. t e t a n i .Specific aerosol precautions are required Tsee below, 111, 3)when large volumes (6 or more l i t e r s ) of biohazardous materialsare centrifuged.Cl-ass I11 Experiments: The same minimal standards described for Class I1 areapplicable with the added provisions that:1. No mouth pipetting of potentially biohazardous material ispermitted. Mechanical pipetting devices are required.2 . The experiments are performed i n laboratories t h a t are undercontrolled access. This does not require a separate room i nwhich no other work is concurrently being conducted. Rather,the i n t e n t of t h i s containment feature is t o exclude extraneouspersons from the area and, hence, reduce the number ofexposed individuals should a laboratory s p i l l or other accidentoccur. Appropriate biohazard signs w i l l be posted on the doorsof laboratories during biohazardous experimentation as w e l l ason the doors of storage areas or cabinets containing potentiallyhazardous materials. Visitors to these work areas are prohibitedunless they have permission from the investigator i n chargewho is responsible for the v i s i t o r s while they are i n the area.3. Specific aerosol precautions are mandatory (see for example,R.L. Dimmick, W.F. Voge and M.A. Chatigny. Potential for accidentalMicrobial Aerosol Transmission in the Biological LaboratoryIn Biohazards i n Biological Research ed A. Hillman, M.N.oxmanand R. Pollack. Cold Spring Harbor Laboratory, 1973,pp. 246-266). Thus, syringes to which the needle is firmlyfixed (e.g. Luer-Lok) should be used. Screw-capped safetyI cups on centrifuge tubes are required when centrifuging biohazardousmaterials. Operation of centrifuges in hoods orother enclosed areas is desirable. Safety equipment to preventthe dissemination of aerosols generated by blending, sonication,centrifugation, e t c . is commercially available (1).Class I V Experiments: The same minimal staniards required £or Class I11 experimentsare applicable with the added provisions that:1. At the minimum, a p a r t i a l containment cabinet (see W.E. Barkly,r e f . 1) or its equivalent should be used for experiments i n t h i scategory. This is a local exhaust ventilation hood with a l i m i -ted front opening in which a i r entering through is subjected tohigh efficiency particulate a i r (Hepa) f i l t r a t i o n or incineratedbefore being exhausted from the area.2. Special aerosol precautions are mandatory for experiments i n t h i sclass. Centrifuges, blenders and other equipment capable ofcreating aerosols should be operated i n separate i s o l a t i o n roomsor hoods (see Dimick, e t . a l . and Bonn, r e f . 1 ) . The standardbiologi c a l hazard s ign used f o r highly i n f e c t i o u s agents (-op. -c i t .p. 1-22) w i l l be posted on cabinets, freezers, r e f r i g e r a t o r s , and/orwork area where biohazardous materials are kept or are beingused. Only personnel who work i n the laboratory may enter thearea when-this sign is posted.Class V Experiments: The potential severity of r i s k entailed i n Class V experimentsd i c t a t e s that they be carried out i n specially constructed f a c i l i t i e s usedto contain highly infectious microbiological agents. In such f a c i l i t i e s , personnelenter through a change room, shower, put on special protective clothing (i.e.disposable gloves, gowns, and foot covers),walk through a disinfecting foot bath,and enter an enclosed laboratory area that containsan "absolute containment cabinet"(Class I11 see Barkly, 2. -c i t . ) . These cabinets are provided with ultrafi l t e r s t h a t can be s t e r i l i z e d , and the hood must be capable of being fumigated.The room i n which the hood is located should be completely sealed, with vaporlocks around the door, l i g h t fixtures sealed into the ceiling, and a l l a i r comingout of the room must pass through appropriate Hepa f i l t e r s . A double door autoclaveshould be mounted i n the wall of the room so t h a t a f t e r s t e r i l i z a t i o n ,materials can be taken out of the autoclave i n t o another room. Upon completionof experimental procedures, the personnel must walk through a foot bath, disposeof clothing which would be s t e r i l i z e d , shower, go back into an entrance room, andput on t h e i r normal s t r e e t clothes.Class V I Experiments: No acceptable level of containment compatible with potentialbiohazard .C. Pertinent References1. Biohazards i n Biological Research. A. Hillman, M.N. Oxman and R.Pollack (ed.). Cold Spring Harbor Laboratory. 1973.2. Manual of.: Clinical Microbiology, 2nd edition. E .H. Lennette , E .H.Spaulding and J.P. Truant (ed.). American Soceity for Microbiology,Washington, D.C. 1974.3. C l a s s i f i c a t i o n of Etiologic Agents on the Basis of Hazard, 3rd edition.U.S. Department of Health, Education and Welfare. Health Services andMental Health Administration, Center for Disease Control, Atlanta,Georgia. June 1972.4. Lab Safety at the Center for Disease Control. Department of Health,Education and Welfare Publication No. HSM 72-8118.5. Handbook of Laboratory Safety, 2nd edition. N.V. Steere (ed.) . TheChemical Rubber Co., Cleveland. 1971.6. Disinfection, S t e r i l i z a t i o n and Preservation. C.A. Lawrence and S.S.Block (ed.). Lea and Ferbiger, Philadelphia. 1971.7. Disinfection. M. Benarde (ed.). Marcel Dekker, Inc., New York. 1970.8. Darlow, H.M. Safety i n the microbiological laboratory. In J . R . Norrisand D.W. Robbins (ed. ) , Methods i n Microbiology. ~ c a d e m i F ~ r e s sI,nc .New York. pp. 169-204, 1969.9. Safety i n Microbiology. D.A. Shapton and R.G. Board (ed.). AcademicPress, Inc., New York. 1972.10. Sulkin, S.E. and R.M. Pike. Prevention of laboratory infections. InE.H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for v i K 1and r i c k e t t s i a l infections, 4th edition. American Public HealthAssociation Inc., New York. pp. 66-78, 1969.11. Chatigny, M.A. Protection against infection i n the microbiologylaboratory-devices and procedures. Advan. Appl. Microbiol. -3:131-192,1961.12. Microbial Contamination Control F a c i l i t i e s . R.S. Bunkle and G.B.P h i l l i p s (ed.). Van Nostrand Reinhold Environmental Engineering Series.Van Nostrand Reinhold Co., New York. 1969.13. Lechevelier, H.A. and M. Solotorovsky. Three Centuries of Microbiology.McGraw-Hill, New York. 1965.I V . RECOMMENDATIONS FOR THE IMPLEMENTATION OF GUIDELINESA. Committees s p e c i f i c a l l y dealing with p o t e n t i a l biohazards associated withrecombinant DNA should be established a t every academic i n s t i t u t i o n and commercialorganization where experiments with p o t e n t i a l or known biohazards areproposed.B. All i n v e s t i g a t o r s wishing to carry out experiments involving possible biohazardswould be required to submit a proposal to the i n s t i t u t i o n a l committee,indicating the purpose of the experiment, the e x p l i c i t b e n e f i t s t o be derived,and an assessment of the p o t e n t i a l biohazards and precautions for containmentt h a t are proposed.C. The r e s p o n s i b i l i t i e s of the committee would be to familiarize themselveswith the extent of p o t e n t i a l biohazards and the necessary measures for t h e i rminimization and containment. It should ensure t h a t no experiments of t h i snature are carried out unless the i n v e s t i g a t o r had submitted such a proposal.It would ensure that the i n v e s t i g a t o r was familiar with appropriate guidelinesand t h a t a thorough review and assessment of the biohazards and t h e i r containmenthad been carried out. It would then evaluate the proposal and anysupporting evidence and would make its recommendation on the proposed research.D. The submitted proposal and the committee's review would be f i l e d as publicdocuments i n a biohazards repository at the i n s t i t u t i o n . This f i l e would besubmitted with a l l grant proposals and applications r e l a t e d t o the experiments.Any subsequent modifications t o the research program which materially affectedthe extent of the biohazards would require a new proposal and a f u r t h e r review.Progress r e p o r t s would be required a t yearly i n t e r v a l s to ensure t h a t theproposed experiments, precautions and containment w e r e adhered to. A completef i l e of all approval programs under study would also be kept i n a federalrepository and would be available for publication. The f i l e of documents oneach proposal would be made a v a i l a b l e by the i n v e s t i g a t o r t o those journalswhere publication policy required appropriate documentation.E. Since the types of experiments under discussion usually require only minimlequipment, of the type generally available a t most academic i n s t i t u t i o n s ,it is recognized that i n the absence of continual supervision or monitoring,the r e s p o n s i b i l i t y t o pursue such a program of research rests f i n a l l y with thei n v e s t i g a t o r . We believe therefore t h a t the P r i n c i p a l Investigator must shoulderthe ultimate r e s p o n s i b i l i t y for the experiments. Thus, r e s p o n s i b i l i t y f o r proceedingwith an experiment should not be s h i f t e d from the PI to a l o c a l committee,absolving the PI from r e s p o n s i b i l i t i e s . The l o c a l committee should make recommendationsand provide advice but cannot approve a program. Thus, even i nface of a favorable review by the committee, an i n v e s t i g a t o r would need toensure t h a t a program s a t i s f i e d the requirements of the guidelines. In theevent t h a t the i n v e s t i g a t o r decided t o ignore recommendations of the l o c a lcommittee, supporting evidence for proceeding with the experiments should beobtained from outside the i n s t i t u t i o n which would j u s t i f y the ultimate courseof action.[We believe that a combination of s c i e n t i f i c i n t e g r i t y combined with peerpressure generated in the face of public a v a i l a b i l i t y and scrutiny of thedocuments mentioned above, w i l l r e s u l t in s t r i c t adherence to the guidelines,w h i l e a t the same t i m e avoiding the extremes of the approval of hazardousseries of experiments by a poorly informed local committee or a veto by alocal committee of experiments which would be generally accepted as validand worthwhile under the conditions of containment that have been proposed.]F. No potentially ecologically hazardous microorganism would be releasedinto the environment intentionally without the approval of an internationalbody which would be duly constituted to make judgements on such release.G. A l l individuals embarking upon experiments categorized as Class I1 toV, should receive training in the handling of potential or infectious materialand must be familiar with the NIH and ASM guidelines (See r e f s 2 and 3; also9, 10 and 11) of experimental use of such materials.[An experimenter who has been w e l l trained in working with pathogenic microorganismsand who is familiar with the ASM Handbook of Clinical MicrobiologyGuidelines should have sufficient expertise to be able to make appropriatejudgements regarding the c l a s s i f i c a t i o n of individual experiments i n thelaboratory situation. Familiarity with t h i s information should enable himto prescribe appropriate containment procedures f o r that particular type ofexperiment and w i l l also enable him to make correct judgement about the typeof training required for technical personnel t h a t may participate i n theexperiment.]H. In those countries where experiments of the type referred to i n t h i sproposal are being carried out, it would seem necessary that national bodiessould be constituted to establish, monitor and promulgate guidelines. Aninternational body should also be established1. to consult with and advise national organizations on t h edevelopment and implementation of guidelines;2. to encourage the maintainance of uniform standards throughoutthe world;.3. to coordinate and periodically review the efficiency anda p p l i c a b i l i t y of international guidelines; and4. to authorize any dissemination into the environment ofnew recombinant types that are l i k e l y to produce s i g n i f i -cant ecological e f f e c t s .V. CONCLUSIONSWe believe that considerable benefits are l i k e l y to r e s u l t from experimentsinvolving the genetic a l t e r a t i o n of microorganisms. The range ofpossible benefits extends from the use of these techniques to add to ourknowledge of basic biological phenomena, t o possible practical applicationsi n the areas of agriculture and medicine.We believe also that a scale of risks exists i n the construction ofgenetically altered microorganisms, and we are uncomfortable about ouri n a b i l i t y t o assess precisely the extent of such r i s k s for many types ofexperiments. However, we believe that the containment procedures describedi n t h i s proposal w i l l reduce any r i s k to laboratory workers and to theenvironment to a level that is acceptably low and which w i l l allow investigatorsto carry out research i n t h i s area. We believe that certainexperiments should presently not be carried out under any circumstances(i.e. Class VI), but that most experiments can be done i f containmentf a c i l i t i e s appropriate to the r i s k are u t i l i z e d .We recommend that specific steps be taken as soon as possible todevelop cloning vehicle-host systems which w i l l further reduce biohazardpotential, w i l l minimize the necessity of elaborate containment f a c i l i t i e s , ,and w i l l obviate judgements which must necessarily be based on l i t t l e or nodata a t the present time. Specifically, we recommend that special sponsoredprograms be i n s t i t u t e d immediately for the development and t e s t i n g of suchsystems. We recommend also the prompt establishment of experimental programsintended to evaluate more f u l l y the potential hazards that may beinvolved i n the genetic a l t e r a t i o n of microorganisms.We believe that perhaps the greatest potential for biohazards involvinggenetic a l t e r a t i o n of microorganisms r e l a t e s to possible military applications.We believe strongly that construction of genetically altered microorganismsfor any military purpose should be expressly prohibited by internationaltreaty, and we urge that such prohibition be agreed upon asexpeditiously as possible.Other recommendations for implementation of the guidelines proposedin t h i s report are contained i n Section IV.APPENDIX ATHE ECOLOGY OF PLASMIDS AND BACTERIOPHAGESA. Plasmid EcologyThe term R plasmid came into use i n 1959 with the discovery i n Japan thatmultiple antibiotic resistance could be transmitted by conjugation from s t r a i n sof r e s i s t a n t epidemic Shigella to drug sensitive Escherichia coli. The significanceof t h i s observation became clear over the next four years when it becameapparent that the problem of transmissible antibiotic resistance was a worldwideproblem with broad implications to medicine. It is now quite clear thatwhen a c l i n i c a l i s o l a t e of an enteric organism displays multiple antibioticresistance, the resistance is most often mediated by an R plasmid. Moreover,recent surveys of the resident f l o r a of asymptomatic individuals i n the community-at-large and the bacteria i n the community environment have shown thatthe incidence of drug resistance has been increasing a t a remarkable r a t e . Theincrease i n the incidence of R plasmids in human populations is, of course,d i r e c t l y linked to the use of antimicrobial agents i n medicine. Domestic animalsalso have shown a parallel emergence of r e s i s t a n t s t r a i n s . However, theentire problem of bacterial drug resistance i n animals is complicated by thefact that most classes of animals grown for food are fed d i e t s containingantibiotic supplements for the stimulation of growth. There has, therefore,been an enormous selection for microorganisms containing plasmids because of amassive a l t e r a t i o n i n the environment.The microbial geneticist was attracted to the study of R plasmids not onlyfrom the standpoint of t h e i r similarity to the c l a s s i c a l F transfer system, butalso from the standpoint of public health, and the unique opportunity tomonitor the extent of change and the genetic basis of change i n natural bacterialpopulations. The increased attention to natural bacterial populations has ledto a broad view of the ecology of bacterial plasmids. For example, fully onethirdof Escherichia c o l i from asymptomatic human and domestic animal populationspossess a t l e a s t one self-transmissible (conjugative) plasmid that confers few orno known phenotypic t r a i t s other than conjugal f e r t i l i t y . Bacterial plasmidsconfer a f a r greater diversity of phenotypic t r a i t s upon the bacteria that possessthem than 'simply' antibiotic resistance or genes (such as enterotoxin biosynthesis)that contribute to bacterial pathogenicity. Plasmids have beenidentified in a variety of bacterial genera and associated with such diversefunctions as the control of lactose fermentation i n Streptococcus -l a c t i s ,sporulation i n Bacillus pumilus, and camphor degradation i n species of Pseudomonas.There has been a growing appreciation of the fact that the genes fora n t i b i o t i c resistance, toxin biosynthesis and other genes such as lactose fermentation,which are of 'transient' evolutionary advantage may be carried byv i r t u a l l y identical molecular vehicles. Thus, it is possible to i s o l a t e conjugativeplasmids which are identical i n over 80 percent of t h e i r molecularlengths but which carry on the one hand a n t i b i o t i c resistance genes, on theother hand genes for the biosynthesis of enterotoxin and, i n yet anotherinstance, genes which control the u t i l i z a t i o n of lactose; there are numerousother examples to suggest that the same plasmid wearing different phenotypicgarb is often isolated independently in several laboratories. To a greatextent it appears that the genetic information which controls essential plasmidfunctions such as replication, the distribution of progeny replicas and,to a somewhat lesser degree, transfer functions is conserved; indeed, plasmids,regardless of phenotype, can be 'speciated' by genetic and molecular studies.However, the origin of and relationships among plasmid genes which determinea n t i b i o t i c resistance and other properties is the subject of considerablespeculation. In some instances, for example certain types of ampicillin andtetracycline resistance, it now appears that the genes i n question reside upona segment of DNA which is readily translocated from replicon to replicon andthat t h i s event can occur i n recombination deficient (E-) bacteria. Morerecent studies have shown that t h i s 'rec' independent translocation of genesis not restricted to antibiotic resistance determinants but a f f e c t s otherplasmid and host genes as w e l l .--- -- -B. Plasmid Transmission i n vivo1. IntroductionGiven a conjugative R plasmid residing within an enteric organism inhabitingthe bowel, the immediate thought might be that R plasmid transfer couldoccur quite readily to other enterobacterial s t r a i n s . This conclusion wouldseem j u s t i f i e d by the laboratory observation that R plasmid transfer from ones t r a i n t o another can usually be demonstrated without d i f f i c u l t y even when itoccurs a t a low rate. The bowel of an animal is a f a r cry from a test tube,however, and from a practical point of view it is important to ask t o whatextent t r a n s f e r occurs -i n -vivo. The a v a i l a b l e evidence i n d i c a t e s t h a t t rans -fer does not occur on so grand a scale i n vivo as it does i n v i t r o . Thefollowing sections which deal specifically with the s t r a i n E. c o l i K-12 outlinethe general parameters of -i n -vivo genet ic t ransmission and wi l l , hopeful ly,help interested investigators to evaluate the steps that must be taken to preventthe dissemination of recombinant plasmid DNA.2. The "Infectivity of E. c o l i K-12"Thus f a r , the 'cloning' of recombinant DNA molecules has been r e s t r i c t e dto substrains of Escherichia c o l i K-12, B or genetic hybrids of the two. BothE. c o l i K-12 and B are long established laboratory s t r a i n s which were i n i t i a l l yisolated from man. One of the f i r s t questions to be asked, therefore, is howcommonly these -E. -c o l i subs t r a ins can colonize t h e human o r animal i n t e s t i n e .Although t h i s precise question has not been studied extensively, it has beenshown that E. c o l i K-12 is a very poor colonizer of the normal bowel. Forexample, a f7e r feeding of between 5 x 10" t o 1x 1012 -E. -c o l i K-12 c e l l s t ocalves, only about 10 c e l l s can be recovered per gram of feces i n 24 hours andby 72 hours cannot be identified a t a l l ( < 1'0 c e l l s ) ( 3 ) . Similarly i n man,ingestion of 10' c e l l s does not normally lead to colonization, indeed, the detectionof more than 100 K-12 cells/gm a f t e r 24 hours is rare. Consequently, itappears t h a t -E. -c o l i K-12 has very l i t t l e inherent capaci ty t o colonize man.There are, however, exceptions to t h i s general rule. If the normal f l o r aof man or animals is disrupted, for example, by therapeutic levels of antibi o t i c s , the inges t ion of -E. -c o l i K-12 bear ing t h e r e s i s t anc e determinants t othese antibiotics leads to colonization a t easily detectable levels (about lo5per gm of feces). Similarly, individuals who have had surgical treatment forstomach or bowel disorders are f a r more easily colonized by a l l enteric species(including E. c o l i K-12). Finally any substance which 'protects' an ingestedorganism from the acidity of the stomach leads often to a higher level of K-12excretion (although subsequent colonization of the normal bowel does not occur,the length of time of excretion may be increased by a few days). Therefore, afew simple r u l e s appear t o be prudent wi th regard t o handl ing -E. -c o l i K-12, partitularlywhen they contain either recombinant DNA molecules or naturallyoccurring plasmids for that matter:a . The usual laboratory procedures employed i n dealing with entericpathogens should be followed as described above.b. Individuals who are receiving a n t i b i o t i c therapy should not workwith the s t r a i n s during the period they are receiving therapy andfor seven days a f t e r the cessation of therapy.- -c. Individuals who have functional i n t e s t i n a l disorders and thosewho have had surgical removal of part of the stomach or bowelshould not work with these s t r a i n s .d. Individuals who take large amounts of antacids should be awarethat they are more readily colonized by ingested bacteria.Obviously, the usual laboratory precaution of no eating i n thelaboratory should be followed.Gene Transfer i n the GutAlthough -E. -c o l i K-12 and B de r iva t ive s do not usual ly a c t i v e l y mul t iplyand colonize the normal animal bowel, the organisms that survive the acidityof the stomach and other natural host defenses, remain viable and can act asgenetic recipients or genetic donors under the proper circumstances. In sofar as we are aware a l l of the recombinant DNA molecules that have been preparedthus f a r are nonconjugative, that is, they do not inherently have thea b i l i t y to i n i t i a t e transfer of DNA. Nonetheless, these nonconjugative plasmidscan be mobilized by a transfer plasmid (such as the classical F plasmid)residing i n the same c e l l . A possible scenario for extension of the reservoirof a recombinant DNA molecule could be as follows: A research worker ingests-E. -c o l i K-12 containing a recombinant DNA plasmid. The surviving c e l l s whilein the gut, engaged i n conjugation with a member of the normal f l o r a containinga transfer plasmid. (Note: about 38%of a l l E. coli s t r a i n s from asymptomaticanimals and man harbor a t l e a s t one transfer piasmid.) The converted K-12 organismcontaining both the transfer plasmid and the recombinant plasmid mates with amember of the normal gut flora and the recombinant plasmid is transferred. Thel a t t e r s t r a i n is f u l l y capable of survival i n the gut and can, i n turn, mate withother s t r a i n s .This hypothetical sequence of events has a certain probability that can becalculated on the basis of laboratory experiments a t 1 i n 10" - 1 i n 10" .perbacterial cell. Experiments of t h i s nature suggest, however, that the probabilitof t h i s occurrence i n the normal gut is on the order of 1i n 10'12 to 1xlo-". This d i f f e r e n t i a l between laboratory and gut i l l u s t r a t e s the conceptthat the best defense against R plasmid and other gene transfer is a normal gutand gut flora. Conditions i n the bowel such as Eh, pH, f a t t y acid concentration,etc. are simply not optimal for genetic transfer. Indeed these same physiologicalconditions of the normal bowel provide us with one of the major naturaldefense mechanisms against infection by enteric pathogens. A major exception is,again, instances i n which the normal f l o r a has been modified by a n t i b i o t i c treatmentor i f there is a functional or pathological bowel disorder. Under thesecircumstances, the r o b a b i l i t y of -i n -vivo t r a n s f e r increases t o an average of1x lo-' to 1x lo-'. Thus, the parameters which affect the colonization ofE. c o l i K-12 likewise affect the probability of genetic transmission and theguidel ines l i s t e d above apply t o t h e prevention of -i n -vivo genet ic transmission.Of course, the probability of gene transfer by an ingested K-12 is exceedinglylow particularly a f t e r the f i r s t 24 hours of ingestion. In our judgement, genetransfer from E. coli K-12 is not a significant hazard so long as normal precautionsof t h e bacteriology laboratory and the containment guidelines l i s t e de a r l i e r are followed.4. Gene Transfer Outside the GutThere is one situation i n which gene transfer might contribute to the disseminationof recombinant plasmid species. This situation could result from an- -- -- -unfortunately common practice i n some laboratories, namely the discarding ofculture supernatants and even viable cultures of E. c o l i K-12 and other "nonpathogenic"bacterial species into the laboratory sink which empties into thecommunity sewer system. On the face of the matter it might be imagined thatv i r t u a l l y any form of sewage treatment would effectively destroy the bacteria..'. This assumption is t o t a l l y unfounded, however. For example, i n Washington,D.C., during periods of heavy water use or during a period of heavy r a i n f a l l ,it is quite possible that a high proportion of organisms disposed of down adz'ain would reach t h e Potomac River where -E. -c o l i counts i n excess of l 0 ~ / 1 0 0m lare not uncommon. (Note that t h i s situation is found, of course, i n most urbanareas). There is relatively l i t t l e data available concerning the frequency ofgenet ic exchange i n water. However, i t i s known t h a t f e c a l -E, -c o l i harboringR plasmids have a very good survival potential i n sewage and i n river water.At any rate, it should be reemphasized that it is not a good practice to disposeof any viable bacterial culture into the community sewage disposal system.This is, of course, particularly c r i t i c a l with respect to cultures containingrecombinant plasmid species or naturally occurring R plasmids for that matter.A l l such s t r a i n s should be considered to have a t l e a s t some minimal degree ofhazard and treated with the common sense experimental practices detailed i nthe section on containment. Similarly, one does not know the potential hazardsof gene transfer on bench tops, etc. which may be contaminated by s p i l l s .Again, one needs to reemphasize the basic methodology that is taught to everybeginning student of microbiology.Roughly 10-15% of normal, asymptomatic individuals harbor E. c o l i andother coloiform organisms i n their nasopharynx. It is not known with anydegree of certainty to what extent well-established laboratory s t r a i n s ofE. c o l i such as K-12 may colonize t h i s anatomical region. This possibilityshould be investigated.References(1) Anderson, J.D., Gillespie, W.A., and Richmond, M.H. (1973) Jour. Med.Microbiology. 6:461-73.(2) Smith, H.W. (19697 Transfer of Antibiotic Resistance from Animal andHuman St r a i n s of Escher ichia c o l i t o Resident -E. -c o l i i n t h e AlimentaryTract of Man. Lancet. i:1174-6.(3) Falkow, S., unpublished experiments.C. Bacteriophage EcologyThe l i t e r a t u r e on bacteriophage is enormous and it would be obviouslyf u t i l e to attempt to summarize a l l that is known about their distribution i nnature. Virulent bacteriophages are capable of only a productive l i f e cyclei n bacteria so that their propagation invariably leads to death and l y s i s ofthe b a c t e r i a l host. Temperate phages on the other hand, as exemplified bythe phage A of E. c o l i K-12, lead a sort of Jekyll-Hyde existence i n bacteria.They are capable of productive growth (lysis) or may become inserted into theb a c t e r i a l chromosome and so assume a r e l a t i v e l y passive role (lysogeny). Thedecision to lyse or insert is under the control of a complex system of geneticallycontrolled biochemical 'switches' and it is possible for the insertedbacteriophage chromosome (called a prophage) to become induced to a productive- -- -- -growth cycle a f t e r peacefully coexisting with the bacterial host for manygenerations. Other temperate phages such as P1, have prophages t h a t do notintegrate into the bacterial chromosome but rather replicate while attachedt o the bacterial inner c e l l membrane. As such, these prophages are plasmids.One need only examine f i l t r a t e s of fecal suspensions, raw sewage, s o i l ,water, unpasteurized diary products or even diseased tissue to learn t h a t bothvirulent and temperate phages are very common i n nature. The systematic searchof bacterial species for the presence of a carried temperate phage is so oftensuccessful t h a t some writers have been moved to remark t h a t it is d i f f i c u l t tobelieve that there are many b a c t e r i a l c e l l s t h a t are not carrying a t l e a s t onetemperate phage! This certainly seems to be the case, for example, whenspeaking of staphylococci but for other b a c t e r i a l species the reported incidenceof carried phage varies from 2% to 94%. Since for the major purposeof t h i s document we a r e pr imar i ly i n t e r e s t e d i n the s t r a i n -E. -c o l i K-12 andthe bacteriophage A and its derivatives, it is probably best t o simply focuson how often E. c o l i species of natural origin carry phages which can alsoi n f e c t -E. -c o l i K-12 and how many of these phages a r e 'lambdoid'.Apparently phages resembling A are not uncommon i n wild-type E. coli.For example some 20 years ago Jacob and Wollman found that 32 or 550 fecal E.c o l i c a r r i e d temperate phages capable of propagation on an -E. -c o l i K-12 de r i -vative. Among these 32 phages, 3 w e r e apparently identical to h and a t l e a s ts i x others could recombine with A. A l l of the other phages could be effectivelycarried by E. c o l i K-12 but were not related to A. More recent unpublishedobservations from several laboratories have confirmed these findings and it isprobably f a i r to say that some 8%to 10% of a l l fecal E. c o l i harbor a t l e a s tone phage capable of i n f e c t i n g -E.-c o l i K-12 and t h a t from 1%t o 2% of f e c a lE. c o l i carry a phage t h a t is closely related to A.Some temperate phages a l t e r profoundly the properties of bacteria thatbecome lysogenized. This process has been termed phage conversion and isresponsible for the synthesis of a number of c l i n i c a l l y important b a c t e r i a lproducts such a s d i ~ h t h e r i at oxin. * (C. - - . . d i ~ h t h e r i a e ) .f i b r i n o l v s i n (S. aureus.) .erythrogenic toxin 13. pyogenes) , tetanus' toxin (c. tetani) , botuli~um toxin(C. botulinum), and for the serological s p e c i f i c i t y of the somatic antigens(endotoxins) of Salmonella species and enteropathogenic -E. -c o l i . I n each case,the bacteriophage genome encodes the genetic information for the synthesis ofthe specific protein product.Phages are capable of transduct ion (phage-mediated gene transfer) and t h i sis probably true for a l l temperate phages as well as some virulent phages.Transducing phages can pick up DNA from prophages and/or plasmids i n donors t r a i n s as w e l l as chromosomal DNA and introduce it into appropriate recipients t r a i n s . Transduction-has been demonstrated to occur in mice by using lysogenicdonor and non-lysogenic r e c i p i e n t s t r a i n s - f o r both S. aureus and -E. -c o l i .Transducing phages or t h e i r DNA are also taken up by ma-lian c e l l s i n culturewhere they p e r s i s t and/or r e p l i c a t e and i n a t l e a s t one instance express functionalgene products.In closing, it should be noted that there has been increasing evidenceover the years to suggest specific relationships between temperate phages andplasmids. Mutant derivatives of A have been found that f a i l to integrate intothe chromosome but replicate and p e r s i s t i n bacterial c e l l s as extrachromosomalDNA or plasmids. The generalized transducing phage pf16 of Pseudomonas putida,i n picking up the genes for degradation of mandelate, was found to acquire thea b i l i t y to a c t as a conjugative plasmid and to promote transfer of bothchromosomal genes and genes for mandelate degradation to recipient strains.The discovery that inheritance of donor genetic markers i n intergeneric matingsbetween E. coli donors and S. typhi recipients and between Klebsiella pneumor@aedonors a<d -E. -c o l i r e c ipi ent s of t en r e s u l t s i n the formation of new plasmids,raises the question as to the origin of the genes to permit autonomous replicationof these elements. The ubiquity of both defective and non-defective prophagesi n lysogenic bacteria that should contain such information leads us tobelieve that such defective and/or non-defective integrated prophages mightcontribute the necessary information for the formation and replication of donorDNA fragments as autonomously replicating circular plasmid molecules i n recipientstrains as a consequence of intergeneric matings.- -- - - -- -- -- - - -APPENDIX BILLUSTRATIVE EXAMPLES OF EXPERIMENTS I N EACH CLASSThe examples given below are mainly for i l l u s t r a t i v e purposes. Some ofthe experiments might not be possible, and there is l i t t l e or no j u s t i f i c a t i o nfor the performance of certain others.A. Examples of Class I Experiment:1. Transductional gene transfer to Escherichia -c o l i using phages P1 orfrom E. c o l i K-12. - - - - - .2. Transformation of E. c o l i K-12 with E. coli K-12 chromosomal, F plasmidor $80 DNA.3. Transformation. transduction. or transfection of Bacillus s u b t i l i s 168wi th B. s u b t i l i s 168 chromosomal D N A ' ~P~BSl phage.4T Transformation of a well-established laboratory s t r a i n of Neisseriacatarrhalis by DNA derived from the same s t r a i n .B. Examples of Class I1 Experiment:1. Conjugal gene transfer between Hfr and F- s t r a i n s of Salmonella typhimuriumLT2.2. Conjugal gene t r a n s f e r between Hfr and F- enteropathogenic -E. -c o l is t r a i n s .3. Formation of a recombinant plasmid between the pSClOl (tetracyclineresistance) and RSFlOlO (streptomycin and sulfonamode resistance) plasmids whenintroduced into E. c o l i s t r a i n K-12.4. Formation of a recombinant replicon between phage X and the ColElplasmid when introduced into E. c o l i K-12.5. Integration of the plasmid R64 into the chromosome of -S. typhimuriumLT2, and its excision to i s o l a t e an R' plasmid.6. A survey of the host range of R plasmids found i n S. typhi s t r a i n sisolated from nature when introduced into - -c o l i K-12, S. Ghimurium LT2E. -and Shigella dysenteriae SH.7. Construction of a recombinant between phage P1 and an ampicillin r e s i s -tance (Ap) plasmid, and the introduction of the recombinant PI-Ap molecule i n t oE. c o l i K-12.8. Construction of a recombinant between bacteriophage Mu and the R plasmidRldrdl9 and its introduction into E. c o l i K-12.9. Construction of recombinant molecules between phage $80 and the Col(Fredericq) plasmid when introduced into - -c o l i . ( I t should be noted that aE.colicin V gene identical or similar to that on the Fredericq plasmid has beenidentified i n a high proportion of bacterial s t r a i n s involved in extra-intesti n a l infection.)10. Construction of a recombinant DNA molecule involving the plasmid of B.pumilus (carrying gene t i c in£ ormation for the inhibition of sporulation) and 2temperate phage from B. s u b t i l i s when introduced into B. s u b t i l i s .11. ~ n t r a ~ e n e r i c t r afnosrm ation of chromosomal DNA i n avi rul ent s t r a i n s ofStreptococci.12. Intrageneric transformation of chromosomal DNA i n Bacillus speciesexcept -B. anthracis.- - -- -- -- -- -- -- -- -- -13. The introduction of bacteriophage X into S. typhimurium.14. Intrageneric chromosome transfer between -E. -c o l i K-12 donor and e i t h e r-S. typhimurium, Proteus mirabilis, of Klebsiella aerogenes recipients.15. Introduction of the genes for nitrogen fixation of the Nif plasmid ofK. pneumoneae into recipient s t r a i n s of E. c o i i K-12.C. Examples of Class I11 Experiment:1. Construction of a recombinant DNA molecule between the cryptic plasmidfrom S. typhimurium LT2 and the Staphylococcus aureus plasmid pI258 and i t sintroauction into S . aureus .2. The introduction of a phage from -S. aureus that leads to production offibrinolysin into a 2. albus s t r a i n .3. Construction of recombinant DNA molecules between sea urchin histonegenes and a - - and t h e i r introducplasmidor bacteriophage replicon from E. c o l i ,tion into E. c o l i .4. Construction of recombinant DNA molecules between the Cm plasmid(specifies chloramphenicol resistance) from -S. pneumoniae and ColE1, and t h e i rintroduction into E. c o l i .5. Construction of a recombinant DNA molecule between X or pSClOl anda plasmid derived from Streptomyces coelicolor and its introduction into E. c o l i .6 . Construction of recombinant DNA molecules between E. c o l i genes involvedi n h i s t i d i n e biosynthesis and a -B. pumilus plasmid, and t h e 2 introduction into-B. s u b t i l i s .7. Construction of a recombinant plasmid or phage that includes fibroingenes from Bombyx mori, when introduced into E. coli.8. Construction of a recombinant DNA moiecule between the chicken ovalbumingene and ColEl and its introduction into E. c o l i .9. Construction of a recombinant moiecule between the OCT plasmid of Pseudomonasputida and e i t h e r phage X or the RSFlOlO plasmid, and its introduction intoE. coli.10. Construction of a DNA chimera between mouse mitochondria1 DNA and phageor the pSClOl plasmid when introduced into E. coli K-12.D. Examples of Class I V Experiment:.1. Construction of recombinant DNA molecules containing DNA from a phage of-S. aureus t h a t codes f o r the production of f i b r i n o l y s i n and e i t h e r -E. -c o l i plasmidor phage DNA, and t h e i r introduction into E. coli.2. Construction of recombinant molecaes between genes for photosynthesis,der ived from any prokaryot ic o r eukaryot ic organism, and -E. -c o l i phage o r plasmidDNA and t h e i r introduction into E. c o l i .3. Construction of a recombinant DNA molecule between plasmid DNA (specifyin g the synthe s i s of kanamycin) from Streptomyces kanamycetic-us and -E. -c o l i plasmidor bacteriophage DNA, and its introduction into -E. -coli.4. Construction of a recombinant between an S. mutans cariogenic plasmidand an -E. -c o l i plasmid and i t s int roduct ion i n t o E, c o l i .5. Construction of a chimeric DNA molecule containing a single purified DNAfragment derived from cucumber mosaic virus and ColEl and its introduction intoE. c o l i .6. Construction of a recombinant between the gene coding for the synthesisof human growth hormone and the pSClOl plasmid, and its introduction into E. coli.- -- -- -- -- -E. Examples of Class V Experiment:1. Construction of a recombinant between the 5. aureus plasmid thatspecifies exfoliative toxin production and an g. coli phage or plasmid, and itsintroduction into E. coli.2. Construction of recombinant DNA molecules between cryptic plasmid DNAfrom microorganisms such as Yersinia pestis, B. anthracis, or Brucella abortusand any other c a r r i e r molecule and t h e i r introduction into E. c o l i .3. Construction of a chimeric DNA molecule which incluhes the DNA of'Dane1 p a r t i c l e s of the h e p a t i t i s B virus and bacteriophage X or plasmid DNA,and its introduction into E. coli.F. Examples of Class V I Experiment:1. Construction of a recombinant between the B phage of Cor~nebacteriumd i p h t h e r i a that specifies toxin production and a phage or plasmid from E. c o l iand its introduction into E. c o l i .2. Construction of a recombinant containing genetic information for toxinproduction from s t r a i n s of Clostridium botulinum or -C. t e t a n i and E. coli phageor plasmid DNA and its introduction into E. c o l i .APPENDIX CGUIDELINES FOR MINIMIZING BIOHAZARDSA. IntroductionI n v e s t i g a t o r s wishing t o construct genetically a l t e r e d microorganismsshould s e l e c t both the DNA cloning vehicle and the r e c i p i e n t s t r a i n with thei n t e n t of achieving the g r e a t e s t possible reduction of known and p o t e n t i a lbiohazards consistent with the a i m s of the p a r t i c u l a r experiment. Wheneverpossible, the i n v e s t i g a t o r s should u t i l i z e a recipient-chimera system designedto (1) minimize possible pathogenicity of the genetically a l t e r e d microorganism;and (2) reduce the likelihood of its dissemination.These goals may be accomplished by s e l e c t i o n of appropriate naturallyoc cur r ing c l ~ n i n gv e h i c l e s and r e c i p i e n t h o s t s , and by s p e c i f i c g e n e t i c manipul a t i o n of these vehicles and hosts. The following suggestions may a s s i s t i ndesign of experiments, and may permit assignment of a p a r t i c u l a r experimentt o a c l a s s i f i c a t i o n having less s t r i n g e n t l e v e l s of containment than mightotherwise be possible. We stress t h a t these i d e a s ' a r e offered as guidelines,and not as requirements, since the d i c t a t e s of any given experiment w i l l determinet o a large extent which, i f any, of these procedures can be u t i l i z e d .B. General Guidance Principles Regarding the Choice of Vehicles f o r DNACloning Experiments1. By s e l e c t i n g and/or genetically manipulating vehicles used i n cloning foreignDNA, i n v e s t i g a t o r s may minimize the possible biohazards involved i n theconstruction of genetically a l t e r e d microorganisms without s a c r i f i c i n g the obje c t i v e s of the experiment. In general, non-conjugative plasmids are preferableto conjugative plasmids as cloning vehicles.2. Cloning vehicles which do not o f f e r any b i o l o g i c a l advantage t o r e c i p i e n tb a c t e r i a a r e preferable to vehicles which may o f f e r such an advantage.3. Cloning vehicles whfch ordinarily have an i n t r a c e l l u l a r existence arepreferable to those e x i s t i n g as encapsulated e x t r a c e l l u l a r p a r t i c l e s .5 . Cloning vehicles t h a t express genotypic or phenotypic properties t h a tare already common i n the r e c i p i e n t b a c t e r i a l species are preferable t o thoseexpressing less common properties.5. A vehicle which has not been subjected t o experimental procedures, suchas mutagenesis, which may a l t e r its b i o l o g i c a l host range, is preferable toa vehicle which has been subjected to such procedures.6 . Cloning vehicles carrying genetic d e f e c t s which may r e s t r i c t t h e i r propagationa r e preferable t o wild-type cloning vehicles.'7. Cloning vehicles that have been w e l l characterized with regard t o t h e i rgenetic and molecular properties are preferable to those which have not beenas well studied.C; Generalprinciples f o r u s e o f AntibioticResistancePlasmids a s c l o n i n gVehicles1. h he cloning vehicle selected must not r e s u l t i n introduction of an a n t i b i o -t i c resistance phenotype to a medically important b a c t e r i a l species i n whichthe r e s i s t a n c e phenotype is not found, e s p e c i a l l yt i c is a drug of choice for the c l i n i c a l control of the species (e.g., i n t r o -duction of p e n i c i l l i n resistance i n t o Streptococcus pyogenes or Streptococcuspneumoneae .)2. The use of plasmids which carry a n t i b i o t i c resistance genes t h a t are normallyr a r e i n extrachromosomal gene pools (e.g. resistance t o trimethoprim and f u s i d i cacid) should be avoided.3. Certain a n t i b i o t i c resistance genes are preferable to others f o r use ass e l e c t i v e agents i n DNA cloning experiments; hence, t e t r a c y c l i n e , sulfonamide,and streptomycin resistance are preferable for use because they occur naturallya t high frequency among microorganisms present i n both human and domestic animalpopulations.Guidelines for Selection of Bacteria as DNA Donors and-Recipients1. Hosts that possess conjugative plasmids or prophages, which may f a c i l i t a t edissemination of genetic material to other hosts, should be avoided i f consiste n t with the objectives of the experiment.2. When l i t t l e is known about the genetic, metabolic, and/or ecological prope r t i e s of a donor or r e c i p i e n t s t r a i n , such s t r a i n s should be avoided forconstruction of genetically a l t e r e d microorganisms.3. Spore-forming microorganisms should not be used as donors or r e c i p i e n t sof chimeric DNA molecules; mutant derivatives unable to form spores should beemployed; r e s t o r a t i o n of sporogeny should not be a possible outcome of theexperiment.E. Suggestions for Possible Genetic Modification of Recipient S t r a i n sGenetic modification of the recipient s t r a i n s p r i o r t o introduction ofrecombinant DNA molecules may contribute f u r t h e r to reducing or eliminatingpossible biohazards. The use of r e c i p i e n t s t r a i n s that possess mutations t h a treduce pathogenicity, a b i l i t y to survive and/or e s t a b l i s h i n a d i v e r s i t y ofecological h a b i t a t s and/or transmit genetic information is therefore desirable.Examplesof g e n e t i c modi f icat ions t h a t can be int roduced i n t o -E. -c o l i s t r a i n s t o accompl i s h the above objectives are provided below:1. Use of a E'mutant since purine-deficient mutants of many pathogenicmicroorganisms are a v i r u l e n t .2. Use of a dap' mutant since the amino acid diaminopimelic acid is notvery prevalent i n n a t u r a l environments and its absence w i l l r e s u l t i n i n a b i l i t yt o synthesize the c e l l wall and thus lead to c e l l l y s i s .3. Use of a temperature-sensitive mutant that cannot grow a t 37OC. Thiswould minimize the a b i l i t y of the genetically a l t e r e d microorganism t o colonizeanimal hosts.4. Use of a cold-sensitive mutant that cannot grow a t temperatures below32°C. This would minimize the a b i l i t y of the genetically a l t e r e d microorganismto survive i n s o i l , water and other n a t u r a l environments.5. Use of a strain that would be unable to ferment or utilize a diversityof carbohydrates- e.g. a pts mutant, phosphotransferase systemdefective. This would contribute to he inability of the genetically alteredmicroorganism to grow in a diversity of ecological habitats.6. Use of a mutant with mutations such as uvr, polA, etc. that wouldconfer increased sensitivity to ultraviolet light, since this would contributeto inability of the genetically altered microorganism to survive innatural environments.7. Use of a rec' mutant since this might reduce the exchange of geneticinformation by themcipient strain.8. Use of a bacterial mutant that is deficient as a recipient of geneticinformation by conjugation. This would reduce the likelihood of introductionof conjugative plasmids from other bacteria in the natural environments andthus reduce the likelihood of mobilization and trailsmission of the informationon the recombinant DNA molecule by conjugation. Some mutations that inhibitconjugation by bacteria may also confer increased resistance to a diversity ofbacteriophages, and thus might reduce the likelihood of transmission of geneticinformation by transduction.9. Use of a mutant that is resistant to a multitude of potential transducingphages since this would minimize the likelihood of dissemination of geneticinformation from the genetically altered microorganism.IAPPENDIX DGUIDELINES FOR MONITORING AND REASSESSMENT OF BIOHAZARDS ASSOCIATED WITHRECOMBINANT DNA MOLECULES INTRODUCED INTO MICROORGANISMSA. IntroductionAfter construction of a recombinant DNA molecule and its introduction i n t oa microbial host, it w i l l be important for the i n v e s t i g a t o r t o assess the r e a lbiohazards associated with the formation of t h i s genetically a l t e r e d microorganism.In many instances the information obtained from these studies w i l l require reclassi f i c a t i o n of the experiment i n t o a new class category. R e c l a s s i f i c a t i o n mightr e s u l t i n the experiment being designated i n a c l a s s requiring l e s s containment,although i n c e r t a i n circumstances the determined biohazards may be moresevere than o r i g i n a l l y expected which would require the r e c l a s s i f i c a t i o n of theexperiment i n t o a c l a s s requiring a more s t r i n g e n t l e v e l of containment.Certain principles should be followed i n obtaining information t h a t mightbe useful i n assessing the r e a l biohazards associated with any given experiment.One should i n i t i a l l y conduct specificexperiments to determine whetherthere a r e any a l t e r a t i o n s i n the pathogenicity of the genetically a l t e r e d microorganismand any changes i n its ecological p o t e n t i a l s . I f the a l t e r e d microorganismcontains DNA specifying unknown gene products it w i l l be d i f f i c u l t ,i f not impossible, t o assess the biohazards associated with the d i s t r i b u t i o n oft h i s genetic information among microorganisms occupying the same ecologicalniches as the r e c i p i e n t s t r a i n . In these instances it w i l l not be possible t or e c l a s s i f y the experiment to employ l e s s s t r i n g e n t degrees of containment. Inthese evaluation experiments, the c e l l s containing recombinant DNA should begrown under the same conditions of containment as were used i n the experimentst h a t produced them. I f c e l l products a r e to be analyzed, the c e l l s should belysed or extracted under these same conditions and these e x t r a c t s tested fors t e r i l i t y prior to taking the material i n t o a general research laboratorywhere l e s s containment is necessary. I f the product is p o t e n t i a l l y toxic, thenappropriate precautions need to be taken t o protect the i n v e s t i g a t o r fromexposure, and s p e c i a l f a c i l i t i e s should be u t i l i z e d to house any animals and/orplants used for t e s t i n g the product. When the genetically a l t e r e d microorganismsare.being evaluated f o r pathogenicity i n animal or plant hosts, these animalsor plants should be under containment f a c i l i t i e s similar to those used f o rthe construction of the genetically a l t e r e d microorganism. Such animal orplant hosts must be disposed of i n a way t h a t w i l l not permit disseminationof the organism being tested. Tests requiring large numbers of a l t e r e d microorganismsshould be avoided i f possible u n t i l there has been some assessment ofthe biohazard. I f t h i s is not possible, then such experiments should be conductedunder conditions of more s t r i n g e n t containment.B. Information That W i l l Be Helpful i n Evalua.ting PathogenicityThe following t e s t s should not be considered to be a l l - i n c l u s i v e since thep a r t i c u l a r t e s t s t o be performed w i l l be dictated by the nature of the genetica l l y a l t e r e d microorganism, with respect to both the origin of the geneticinformation on the recombinant DNA molecule and the p a r t i c u l a r a t t r i b u t e s of therecipient host species. The design and conduct of specific experiments toevaluate the r e a l biohazards w i l l therefore require c a r e f u l evaluation by theinvestigator. Some of the relevant types of experiments that can be conductedon the genetically altered microorganism include determination of itsproperties in the following tests:1. Infectivity in appropriate animals or plants.2. Colonization in the gut, oral cavity, on the skin, etc. ofmodel animal hosts or on the roots, leaves, etc. of appropriateplants.3. Production of keratoconjunctivitis in guinea pigs (theSereny test) which would be an indication of the capacityof the altered microorganism to penetrate the intestinalmucosa .4 . Invasion and proliferation in macrophages and/or fibroblasts.5. Production of such cell products as bacteriocins, hemolysins,fibrinolysins, collagenases, pectinases, etc. that might contributeto colonizing ability and/or invasiveness and toxinsof various sorts and to test the potency of such toxins byusing appropriate cell cultures of eukaryotic organisms,ligated intestinal loops of appropriate animal hosts orappropriate plant or animal species.6. Production of hypersensitivity and/or necrosis by cells orextracts when injected intradermally into the skin of appropriateanimal hosts.7. Determination of the minimal inhibitory concentrations ofvarious antimicrobial agents useful in killing and/or inhibitinggrowth of the altered microorganism.8 . Determination of whether or not the gene products specifiedby the recombinant DNA appear extracellularly, intracellularlyor in the periplasmic space.C. Information That Will Be Helpful in Evaluating Ecological PotentialThe individual experiments needed to assess ecological potential of thealtered microorganism will of necessity be dictated by the properties of the -strains used to construct it. The following types of experiments should thereforeonly serve to illustrate the range of tests to determine the propertiesof the genetically altered microorganism:1. Expression of the genetic traits that are specified by therecombinant DNA molecule.2. Resistance to W,disinfectants, etc.3. Survival in soil, water and the dry state or in any ecologicalhabitat likely to be occupied.4. Ability to form spores.5. General metabolic activities and attributes includingchanges in growth rate, utilizable and preferred substrates,temperature and pH optima for growth, aerobic vs. anaerobicgrowth, photosynthetic and N2 fixing ability, etc.6. Production of substances that displace or inhibit other microorganismsthat normally occupy the same ecological habitats.D. Other Information Needed to Evaluate the Severity of BiohazardsIt will be extremely important to test the ability of the recombinant DNAcontained in the altered microorganism to be transmitted by phage and/or-- --conjugative plasmids to other s t r a i n s of the same species as the recipient aswell as to other species of bacteria known to exchange genetic information withthe recipient host species. Such t e s t s should also be performed with others t r a i n s of the bacterial species from which DNA was obtained to construct therecombinant DNA, even when these species are not known to exchange geneticinformation with each other. Since some microbial species are known to excreteDNA into the medium which is sometimes biologically active, t e s t s should alsobe done to determine whether the recombinant DNA is capable of being taken upand expressed i n other microorganisms by transformation. Such t e s t s forexamining transmission of the recombinant DNA by transduction, transfection,conjugation, transformation and/or by encapsulation of the recombinant DNA i nphage virions should be tested in v i t r o experiments and in some instances underin vivo conditions with appropriate animal and/or plant hosts.E. SummaryIf one performs any or a l l of the above experimental t e s t s to evaluatepotential biohazards of genetically altered microorganisms, it w i l l be necessaryto include as controls the organisms used as donors of the genetic informationto form the recombinant DNA molecule as well as the recipient hoststrain.Dr. Donald R. HelinskiDepartment of BiologyUniversity of CaliforniaF.O. Box 109La Jolla, California 92037Dear Don :I have had the opportunity to read over the s m a r y statement of the organizingcommittee for the hilomar Conference, and I share your concern and dismay.According t o the way the statement reads, one is free to tranefer regions of oncngenicv i r a l DNAs into g. coli with "safer" vector host system tinder low risk containment,but i f one were to introduce metabolism genes from a sporulation deficients t r a i n of 15. subtllus into ,g. colf which "can confer upon the recipientorganisms new metabolic properties not native to the species", one would requiremoderate or high risk containment --- apparently regardless of what cloning vehiclesor recipient c e l l s t r a i n s are used. Similarly, moderate or hiah risk contafnmentmust be used "if prokaryotic experiment8 involve pat!~ogenic organisms"(how ambiguous! What does "involve" mean? Is E. coli a "pathogenic organism"?),but as soon as an animal virologist concludes that he has a "safer vector-hostsystem':, he w i l l be able to free1.y introduce "characterized" segments of pathogenicanirnal viruses into bacteria under low r i s k containment.Xnte that the report does not define what is an acceptably "safer" vectorhostsystenl, nor does it require any testing of the safety of the system, exceptfor high risk experiments. I s the development of temperature sensitive ColEl orpSClOl plasmids or a UAP-raquiring E. coli mutant going t o be judged sufficientto allow cloning of pathogenic v i r a l genomes in bacteria under low-risk containment?Cre disparity between the level of safety required by the document forprokaryotic genes and that requfred for animal virus genes is astonishing.It is also interesting to note that once cloned segments of even wannbloodedvertebrate DNA are completely characterized, they no longer need bemaintained in moderate r i s k containment laboratories. Ibwever, no such allowanceis provided for non-pathogenic prokaryotic genes once they have been characterized.'ke statement a t the bottom of page 4 requiring the adoption of "safer" vectorsand hosts as they become available needs to he modified. It implies thatexisting vectors are inadequate for low-risk experiments. The use of newer vectorsfor low-risk experiments may be fine i f all you are interested in is theforeign gene being cloned, but many low r i s k experiments involving prokaryoticgenes u t i l i z e a particular vector as an integral part of the experiment --- andtheree4uirement to adopt another one may change the experiment conceptually. Theinvestigator must be given f l e x i b i l i t y for low r i s k studies.It 5s interestfng ( ~ a g c3) that capaclty ro introduce new netabolfc activiti e s is not even considered i n the case of eukaryotfc ox animal v f r a l DNA, buton1.p pathogenicity is. 1To~iever. this point is considered for prokaryotic studies.An2 f'shotgunll experiment from a prokaryot ic organism having ~netabol ica c t i v i t i e s found i n .:-.-not &has the potential of conferring these new a c t i v i t i e s on someof the transformants. Tlie document would require moderate r i s k containment forsuch experiments. Y e t , although the sane concern is valid for shotgun experfmentsinvolving DNA from eukaryotic organisms (which certainly contain metabolic activi t i e s not founcl in 2. &I, these shotgun experiments would require only lowr i s k containment.I wonder what evidence there is for the statement on page 10 of the documentthat "genetic transformation of bacteria does occur i n animalst1. Also, page 5,line 2, "possibility" should certainly be substituted for "probability". Also,page 3, item 4, should read "Class I11 and hlghers'.Xy overall view of the docume?tt is that it makes light of the dangers whichmay result fron recombinant DNA molecule experiments involving oncogenic and otherpathogenic v i r a l genomes, while posing restrictions that prevent introduction ofs i m l e metabolism genes from other species into &. &. As I read the document,it would even make no difference i f the genes could be put into 2. coli from theother species by conjugation or transduction, moderate or high r i s k containmentwould be required so long as the genes are not "native" to the recipient species".At: this noint, Don, 1 have had it. I have spent a major amount of my timefor more than a year attempting t o contribute whatever I could to the developnentof c r ~ d i b l e , internally consistent, and appropriate guidelines that would insuresafety of experimentation i n genetic m i p u l a t i o n . We now appear to have recommendationsdesigned to meet the specific experimental needs of animal virologists(the explicit reduction of containment level required for "demonstrably non-transfomlnp,regions of oncogenic v i r a l DNA" is almost funny:), while the considerationof prolcaryotfc experiments uses entirely different standards.In this l e t t e r I have pointed out the specific itema that lead to my mostserious concern. I hope that these comments w i l l be useful to you and to theother members of the WIH comlttee in attempting to formulate a meaningful codeof practice for the conduct of experiments i n t h i s area.I w i l l be leaving for England in several weeks, and boy an I gihad! Pleasetelephone me a t Stanford i f you would like to discuss any of the specific pointsi n t h i s l e t t e r .With best wishes,Sincerely yours,Stan CohenSNC :lrc.c. : Stan Falkow, Roy Curtiss, Dick Novick, Roy ClowesNature Vol. 261 June 24 1976to be patented?A patent application governing commercialuses of recombinant DNAtechniques has been filed in the UnitedStates. Colin Normun reports.WHILE the scientific community hasbeen loudly debating the potentialhazards and benefits associated with anew technique for manipulating genesin living organisms, Stanford Universityand the University of Californiahave ,been quietly trying to patent thetechnique in the United States.Rumours about the patent application,which apparently has been pending forat least 18 months, surfaced at a scientificsymposium held at the MassachusettsInstitute of Technology earlierthis month; they were confirmed byuniversity officials last week.Though the people concerned withthe patent application are reluctant todiscuss it in detail while the matter ispending, the patent is understood to beworded broadly enough to cover commercialuses of any method of transplantinggenes from one organism intoanother. The patent, if awarded, wouldnot affect research uses of the technique,and it would apply only in theUnited States.The basis for the application is researchpublished in 1973 and 1974 bygroups led #by Stanley Cohen at Stanfordand Herbert Boyer at the Universityof California, San Francisco. Theirefforts led to the first demonstrationthat genes can be snipped from theDNA of virtually any organism andspliced into a bacterial plasmid (a ringof DNA which reproduces inside abacterium independent of the bacterium'schromosomes). The key par.tof their research showed that the modifiedplasmid could be reinserted into abacterium so that the transplanted,'foreign' genes are copied each timethe bacterium reproduces. It is understoodthat the patent applicationcovers the process for constructinghybrid DNA moleoules capable ofself-reproduction, which means that, ifawarded, it would also apply to transplantinggenes into viruses and bacteriophages.There has been speculation that thetechnique might, eventually, lead toGenetic manipulationsuch commercial uses as the insertioninto bacteria of genes capable of producingpharmaceutical products, suchas insulin, so that specially engineeredbacteria would be capable of secretingexpensive drugs. Another speculativecommercial application would be tograft genes capable of fixing nitrogeninto crop plants such as wheat, to producea new variety capable of synthesisingits own nitrogen fertilisers fromthe atmosphere. Such uses of the technique,would be covered by the patentapplication.Discussion of the patent applicationarose at a symposium on genetic manipulationheld by Miles Laboratories atMIT on June 8-10. Noting that therehave been persistent rumours thatsomebody is trying to patent the technique,a speaker asked whether anyparticipants could shed some light onthe matter. Cohen, who was present atthe meeting, confirmed that Stanfordand the University of California arelooking into the possibility of takingout a patent, and he emphasised thatneither he nor any of the other researchersinvolved would benefit financiallyif it were awarded.A patent application can be filed inthe United States up to a year afterthe information on which it is basedhas been made public. The first paperby Cohen and Boyer's groups was publishedin November, 1973, which indicatesthat the application should havebeen filed before November 1974.Asked last week whether that assumptionis correct, a Stanford patentofficer replied that "it would be reasonableto assume that we were prudentin filing our application". In some countries,such as Great Britain, a patentmust be applied for before any publicdisclosure is made. Britain's NationalResearch and Development Corporationinitially looked into the possimbilityof seeking a patent on genetic manipulationtechniques developed by Kennethand Noreen Murray at EdinburghUniversity, but dropped the idea becauseof prior disclosure.When the matter was raised at theMiles symposium, two concerns werediscussed. First, some scientists wereworried that the patent, if awarded,might interfere with research. Andsecond, it was suggested that it mayforce some commercial concerns toseek a less safe, but patent-free, geneticmanipulation technique. Neither concernseems to be valid, however, andin fact the patent, if awarded, couldhave some safety benefits.As for the research implications, thepatent would apply only to commercialuse of the technique-it would notcover either academic or industrialresearch uses. Moreover, asked whetherit may hold up beneficial applicationsof the technique, a Stanford officialargued that royalties derived from thepatent would be "reasonable" andwould not limit its use.As far as safety implications are concerned,the application seems to coverthe key steps in the genetic manipulationprocess and thus, unless it isdrastically narrowed by the US PatentOffice, it would be difficult to see howa different patent-free process couldbe developed. If the patent is awarded,Cohen suggested at the Miles symposiumthat Stanford and the Universityof California could insist that commercialusers of the process be requireddo sign an undertaking to abideby safety guidelines laid down by theNational Institutes of Health (NIH).NIH is due to issue guidelines onJune 23 governing genetic manipulationresearch which it supports. At ameeting earlier this month, NIHDirector Donald Frederickson briefeda number of industry officials on theguidelines, and though they met withgeneral support, some drug companyrepresentatives expressed reservationsabout one or two provisions. The NIHguidelines, moreover, would not bebinding on industry, since they wouldapply only .to NJH-sponsored research.The ,patent may, therefore, providea means of ex,tending .the coverageof the guidelines.Cohen and Boyer's work was supportedby the National Institutes ofHealth, but the federal government isunlikely to have a stake in the patent.Stanford has a standing agreementwith the Department of Health, Educationand Welfare (HEW) which givesthe university patent rights on inventionsproduced from research supportedby HEW grants, unless the project hasbeen exempted from the agreement.According to an HEW official, thegenetic manipulation studies were notexempted. Stanfotd patent officers have,however, been discussing the applicationwith federal officials, particularlyas regards establishing licensing arrangementsif the patent is awarded. 0DEPARTMENT OF HEALTH, EDUCATION, AND WELFAREPUBLIC HEALTH SERVICENATIONAL INSTITUTES OF HEALTHBETHESDA. MARYLAND 20014August 23, 1976Dr. Stanley N . CohenDepartment of MedicineStanford University Schoolof MedicineStanford University Medical CenterStanford, CA 94305Attention: Diane E. BushDear Dr. Cohen:Enclosed are the NIH Guidelines for Research Involving RecombinantDNA Molecules and the Decision of the Director, NIH, as publishedin the Federal Register.The next printing of the Guidelines w i l l be in a manual style whichw i l l facilitate periodic updating. In the near future, a postcardw i l l be sent to a l l concerned to determine those who wish to receivethisformat.information is needed, please feel free to contact me.Sincerely yours,. IrGAC:kcb;"/ Acting Diirreeccttoorr vOrfrflicce ooff Recombinant DNA Act ivi t iesNa-tio. n al Ins t i tut e of General M e d i ~ lI- -RECOPBINANT DNA: FACT AND FICTIONStanley N. CohenStanford University School of MedicineStatement prepared for a meeting of the Committee onEnvironmental Health, California Medical AssociationNovember 18, 1976Almost three years ago, I joined with a.group of s c i e n t i f i c colleaguesi n publicly c a l l i n g a t t e n t i o n to possible biohazards of c e r t a i n kinds ofexperiments t h a t could be carried out using newly developed techniques forlinking together genes from diverse sources i n t o biologically functionallymolec,ules. Because of the newness and r e l a t i v e simplicity of these techniques,we were concerned t h a t experiments involving c e r t a i n genetic combinations t h a tseemed to us to be hazardous might be performed before adequate consideration hadbeen given t o the p o t e n t i a l dangers. Contrary to what was believed by many observers,our concerns pertained t o a few very s p e c i f i c type of experiments t h a tcould be c a r r i e d out using the new techniques,not to the techniques~themselves.Guidelines have long been available to p r o t e c t laboratory personnel andthe general public against known hazards associated with the handling ofc e r t a i n chemicals, radioisotopes, and pathogenic micro-organisms , but becauseof the newness of recombinant DNA techniques no gbidelineswereyet available f o r t h i sresearch. My colleagues and I wanted to insure t h a t the powerful new techniqueswouldn't be employed, for example, for the construction of Streptococci~r Pneumococci r e s i s t a n t to p e n i c i l l i n --- or f o r the creation of E. c o l icapable of synthesizing botulinum toxin or d i p t h e r i a toxin. We asked t h a t theseexperiments not be done, and also called for d e f e r r a l of construction ofrecombinants containing tumor v i r u s genes u n t i l the implications of such experirt ments could be given f u r t h e r consideration.During the past two years, much f i c t i o n has been written about "recombinantDNA research".. What began as an a c t of r e s p o n s i b i l i t y by s c i e n t i s t s ,faeluding a number of those involved i n the development of the new techniques,has become the breeding ground f o r ahorde.of p u b l i c i s t s --- most poorlyinformed, some well-meaning, some self-serving. This statement represents anattempt to i n j e c t some relevant f a c t s i n t o the extensive public discussion ofrecombinant DNA research. F i r s t of all:I tR ecombinant DNA research" i s n o t a s i n g l e e n t i t y , b u t r a t h e r i s a technique .t h a t can be used f o r a wide varie.ty of possible experiments. Much confusion--has resulted from a lack of understanding of t h i s point by many who have writtenabout the subject.. Recombinant DNA techniques, l i k e chemicals on a s h e l f , aren e i t h e r "good" nor "bad" per se: c e r t a i n experiments t h a t can be done usingthese techniques are l i k e l y to be hazardous ( j u s t as c e r t a i n experiments donewith chemical combinations taken from the shelf w i l l be hazardous), and there isuniversal agreement t h a t such recombinant DNA experiments should not be done.Other experiments t h a t use the very same techniques, such as taking apart a DNAmolecule and putting segments of it back together again, are without conceivablehazard -- and anyone who has looked i n t o the subject has concluded t h a t theseexperiments can be done without concern.Then, there is the area "in between" : for many experiments, there is noevidence of biohazard, but there is also n o c e r t a i n t y t h a t there is not a hazard.For these experiments, guidelines have been developed i n an attempt to match al e v e l of containment with a degree of hypothetical r i s k . Perhaps the singlepoint t h a t has been most misunderstood i n t h e controversy about recombinant DNAresearch, is t h a t discussion of "risk" i n the middle category of experimentsr e l a t e s e n t i r e l y t o hypothetical and speculative p o s s i b i l i t i e s , not expectedconsequences o r even phenomena t h a t seem l i k e l y t o occur on the basis of whatis known. Unfortunately, much of the speculation has been interpreted as f a c t .There is nothing novel about the p r i n c i p l e of matching a l e v e l of concainmentwith the l e v e l of anticipated hazard; t h e containment procedures used f o rspathogenic b a c t e r i a , toxic substances, and radioisotopes attempt t o do t h i s .However, the containment measures used i n these areas address themselves onlyt o known hazards and do not attempt to p r o t e c t against the unknown. I f thesame p r i n c i p l e of protecting only against known hazards were followed i n recombinantDNA research, there would be no containment whatsoever except f o r a veryfew experiments. In t h i s instance, we are asking not only t h a t there be noevidence of hazard, but t h a t there be p o s i t i v e evidence t h a t there is no hazard.In developing guidelines f o r recombinant DNA research we have attempted to takeprecautionary steps to protect ourselves against hazards which are not knownt o e x i s t -- and t h i s unprecedented act of caution is so novel t h a t i t has beenwidely misinterpreted a s implying the imminence or at l e a s t the likelihood ofdanger.Much has been made of the f a c t t h a t even i f a p a r t i c u l a r recombinant DNAmo1eciL.e shows no evidence of being hazardous at the present time, we areunable to say f or^ c e r t a i n t h a t it w i l l not devastate mankind some years hence.Of course t h i s statement is c o r r e c t , j u s t as we are unable t o say f o r c e r t a i nt h a t the vaccines we are administering to millions of children do not containgenes t h a t w i l l produce contagious cancer some years hence, we a r e unable t osay f o r c e r t a i n t h a t a huge meteor w i l l not f a l l on and destroy New York Citynext week, we a r e unable t o say f o r c e r t a i n t h a t a new v i r u l e n t v i r u s w i l l nota r i s e spontaneously next winter and cause a world wide f a t a l epidemic of ah i t h e r t o unknown disease, we a r e unable t o say f o r c e r t a i n t h a t novel hybridplants being bred around t h e world w i l l not suddenly become weeds t h a t w i l lovercome our major food crops and cause world wide famine, e t c . , e t c . , e t c .The statement t h a t p o t e n t i a l hazards could r e s u l t from c e r t a i n experimentsusing recombinant DNA techniques is akin t o the statement t h a t a meteor couldwipe out New York City next week, a world wide pandemic caused by a new mutantv i r u s could destroy mankind, a vaccine i n j e c t e d today i n t o millions of peoplecould lead to cancer i n twenty years or a new plant species could uncontrollablydestroy the world's food supply. W e have no reason t o expect t h a t any of thesethings w i l l happen, but we a r e unable t o say f o r c e r t a i n t h a t they w i l l nothappen. Similarly we a r e unable to guarantee t h a t any of man's e f f o r t s toinfluence the e a r t h ' s weather, explore space, modify crops, o r cure disease w i l lnot carry with them the seeds f o r the ultimate destruction of c i v i l i z a t i o n . Canwe i n f a c t point t o one major area of human a c t i v i t y where one can say f o r c e r t a i nthat there is zero risk? Clearly, it is possible t o develop plausible "scarescenarios" involving v i r t u a l l y any a c t i v i t y o r process, and these would haveas much (or as l i t t l e ) basis i n f a c t as most of the scenarios involving recombinantDNA. We must distinguish f e a r of the unknown from f e a r t h a t has somebasis i n f a c t ; t h i s appears t o be the crux of the controversy surroundingrecombinant DNA.Unfortunately, the public has been led t o believe t h a t the biohazardsdescribed i n various scenarios are l i k e l y or probable outcomes of recombinantDNA research. "If the s c i e n t i s t s themselves are concerned enough to r a i s e theissue", goes the f i c t i o n , "the problem is probably much worse than anyone w i l ladmit." However, the simple f a c t is t h a t there is no evidence to even suggest t h a ta bacterium carrying any recombinant DNA molecule poses a hazard beyond thehazard t h a t can be predicted from the known properties of the components of therecombinant. And experiments involving genes from organisms carrying genes t h a tproduce t o x i c substances or pose other known hazards are prohibited.Freedom of S c i e n t i f i c InquiryThis issue has been raised repeatedly during discussions of recombinantDNA research. "The time has come" the c r i t i c s charge, "for s c i e n t i s t s t o abandont h e i r long-held b e l i e f t h a t they should be f r e e to pursue the a c q u i s i t i o nof new knowledge regardless of the.consequences". The f a c t is t h a t no one hasproposed t h a t freedom of inquiry should extend to s c i e n t i f i c experiments t h a tendanger public safety. Yet, "freedom of s c i e n t i f i c inquiry" is repeatedlyraised a s a s t r a w man i s s u e by c r i t i c s who imply t h a t somewhere there are thosewho argue t h a t there should be no r e s t r a i n t on research.Instead, the history of t h i s issue is one of self-imposed r e s t r a i n t bys c i e n t i s t s from. the very s t a r t . The s c i e n t i f i c group t h a t f i r s t raised thequestion of possible h a z a r d . i n some kinds of recombinant DNA experimentsincluded most of the s c i e n t i s t s involved i n t h e development of the techniques --and their,concern was made public so t h a t other i n v e s t i g a t o r s who' had not adequatelyconsidered the p o s s i b i l i t y of hazard could exercise appropriater e s t r a i n t . While most s c i e n t i s t s would defend t h e i r r i g h t to freedom ofs c i e n t i f i c thought, and discourse; I do not know of anyone who would proposet h a t s c i e n t i s t s should be f r e e to do whatever experiments they choose regardlessof the consequences.Interference with "evolutionary wisdom"We a r e asked by some c r i t i c s of recombinant DNA research to believe t h a tthe process of evolution of plants and animals has remained d e l i c a t e l y controlledfor millions of years, and t h a t the construction of recombinant DNA molecules nowthreatens the master plan of evolution. Such thinking, which requires a b e l i e ft h a t nature is endowed with wisdom, i n t e n t , and f o r e s i g h t , is a l i e n t o mostpost-Darwinian b i o l o g i s t s . Is there any evidence t h a t the evolutionary process ispresently delicately contrzdled by nature? To the contrary, man has long agomodified the process o f ' e v o l u t i o n as it existed i n the Garden of Eden, and: biological evolution continues to be dominated by man. Primitive man's domesti c a t i o n of animals and c u l t i v a t i o n of crops provided an "unnatural" advantageto c e r t a i n biological species and a perturbation of evolution. The l a t e rcreation by man of hybrid plants and animals has resulted i n the propagationof new genetic combinations t h a t are not t h e product of n a t u r a l evolution. Inthe microbiological world, the use of antimicrobial agents to t r e a t infectionshas i n t e r f e r e d i r r e v e r s i b l y with any d e l i c a t e evolutionary control t h a t mayhave existed previously.In a recent letter t o . Science t h a t has been widely quotedby c r i t i c s of recombinant DNA research,Professor Erwin Chargaff of Columbia University asks, "Have we t h e r i g h t t ocounteract i r r e v e r s i b l y the evolutionary wisdom of millions of years . . .?I1 Itis t h i s so c a l . l e d ' ~ v o l u t i o n a r y wisdom'' t h a t gave us the combinations f o rbubonic plague, smallpox, yellow fever, typhoid, polio, diabetes, and f o r t h a tmatter -- cancer. It is t h i s "wisdom" t h a t continues t o give us uncontrollablediseases such as Lassa fever, Marburg v i r u s , and very recently the Marburg-relatedhemorrhagic fever virus which has resulted i n nearly 100% m o r t a l i t y i n i n f e c t e dindividuals i n Zaire and the Sudan. The acquisition and use of all b i o l o g i c a land medical knowledge c o n s t i t u t e s an i n t e n t i o n a l and continuing a s s a u l t on"evolutionary wisdom". Is t h i s the- "warfare against nature" t h a t ProfessorChargaf f f e a r s from recombinant DNA?How about the "benefits?"For a l l but a very few experiments, the "risks" of recombinant DNA researchare e n t i r e l y speculative and it seems appropriate t o ask those who claim otherwisefor evidence t o support t h e i r a s s e r t i o n s . Are t h e ' b e n e f i t s equally specula t i v e or is there some f a c t u a l basis f o r expecting t h a t b e n e f i t s w i l l occurI believe t h a tfrom t h i s technique? the a n t i c i p a t i o n of b e n e f i t s has a s u b s t a n t i a l basis i n f a c t ,a n d . t h a t the benefits f a l l i n t o two p r i n c i p a l categories: (1) advancementof fundamental s c i e n t i f i c and medical knowledge, and ( 2 ) possible p r a c t i c a lapplications.I n the s h o r t space of three and a half years the use of the recombinantDNA technology has already been of major importance i n the advancement offundamental knowledge. We need t o understand t h e s t r u c t u r e and functionof genes and t h i s methodology provides a way t o prepare l a r g e q u a n t i t i e sof s p e c i f i c segments of DNA i n pure form. For example, recombinant DNAmethodology has provided us with much information about the s t r u c t u r e ofplasmids t h a t cause a n t i b i o t i c r e s i s t a n c e i n b a c t e r i a , and has given usi n s i g h t s i n t o how these elements propagate themselves, how they evolve, andhow t h e i r genes a r e controlled. I n the past, our i n a b i l i t y t o i s o l a t e s p e c i f i cgenetic regions of the chromosomes of higher organisms has l i m i t e d our understandingof the genes of complex cells; now with preparations of pure genes wecan begin t o examine the s t r u c t u r e and function of normal andmalfunctioning chromosomes,t o l e a r n for example how abnormal hemoglobins a r e produced i n c e r t a i nhuman disorders. Use of recombinant DNA techniques has provided important newinsights .into how genes are organized i n t o chromosomes and how gene expressionis controlled. With such knowledge we can begin to learn how defects i n thes t r u c t u r e of such genes a l t e r t h e i r function and re.gulation.On a more p r a c t i c a l l e v e l , recombinant DNA techniques p o t e n t i a l l y enablethe construction of b a c t e r i a l s t r a i n s that can produce biologically importantsubstances such as antibodies and hormones. Although the f u l l expression ofhigher organism DNA t h a t is necessary t o accomplish such production has notyet been achieved i n b a c t e r i a , the s t e p s that need to be taken t o reach t h i sgoal are defined, and we can reasonably expect t h a t the introduction of appropr i a t e " s t a r t " and ' I s top" control s i g n a l s i n t o recombinant DNA molecules w i l lenable the expression of. animal c e l l genes. On an even s h o r t e r time s c a l e , wecan expect recombinant DNA techniques to revolutionize the production of a n t i -b i o t i c s , vitamins, and medically and i n d u s t r i a l l y useful chemicals by eliminatingthe need to grow and process the often exotic b a c t e r i a l and fungal s t r a i n scurrently used as sources &or such agents. We can a n t i c i p a t e the constructionof modified antimicrobial agents t h a t are not destroyed by the a n t i b i o t i ci n a c t i v a t i n g enzymes responsible for drug resistance i n b a c t e r i a .In the area of vaccine production, we can a n t i c i p a t e the construction ofs p e c i f i c b a c t e r i a l s t r a i n s able to produce desired antigenic products --eliminating the present need for immunization with k i l l e d o r attenuated specimensof disease-causing viruses. One p r a c t i c a l application of recombinant DNAtechnology i n the area of vaccine production is already close to beingrealized: an E. co-li plasmid coding f o r an e n t e r i c toxin f a t a l to livestockhas been taken a p a r t , and the toxin gene has been separated from the remainderof the plasmid. The next s t e p is to cut away a small segment of the toxinproducinggene so t h a t the substance produced by the r e s u l t i n g gene w i l l n o t .have toxic properties but w L l l be imunologically a c t i v e i n stimulating antibodyproduction.Other b e n e f i t s from recombinant DNA research i n the areas of food andenergy production a r e more speculative. However, even i n these areas thereis a s c i e n t i f i c basis for expecting t h a t the 'benefits w i l l someday be r e a l i z e d .limitedThehavailability of f e r t i l i z e r s and the p o t e n t i a l hazards associated with excessiveuse of f e r t i l i z e r s presently l i m i t s c e r t a i n crop yields, but a g r i c u l t u r a lB e e f t s suggest € R a t trafieglantation of the nitrogenase system from theCht=c%a@emes uf eef€eifi be€teria i n t o plants o r i n t o other b a c t e r i a t h a t l i v esymbiotically with food crop plants may eliminate the need for f e r t i l i z e r s .For many.years, s c i e n t i s t s have modified the heredity of plants by comparati v e l y primitive techniques. Now there is an e f f i c i e n t means of doing t h i sby design, rather than by chance.Certain algae are known t o produce hydrogen from water using sunlight asenergy. This process p o t e n t i a l l y can yield a v i r t u a l l y l i m i t l e s s source ofpollution-free energy i f technical and biochemical problems indigenous t o t h epresently known hydrogen-producing organisms a r e solved. Recombinant DNAtechniques represent a possible means of s o l u t i o n of these problems.Even i f hazards are speculative and t h e p o t e n t i a l b e n e f i t s are signi f i c a n t and convincing, wouldn't it still be b e t t e r t o carry out recombinantDNA experiments under conditions t h a t provide an added measure of s a f e t y --j u s t i n case some of the conjectural hazards prove to be r e a l ?This is exactly what is required under the NIH guidelines for recombinantDNA research :1. These guidelines prohibit experiments i n which t h e r e is some s c i e n t i f i cbasis f o r a n t i c i p a t i n g t h a t a hazard w i l l occur. In addition, they prohibitexperiments i n which a hazard, although it might be e n t i r e l y speculative, wasjudged by the National I n s t i t u t e s of Health t o be p o t e n t i a l l y serious enoughto warrant prohibition of the experiment.2. The guidelines require t h a t a large class of other experiments be c a r r i e d-out i n P4 high l e v e l containment f a c i l i t i e s of t h e type designed f o r work withthe most hazardous naturally-occurring microorganisms known t o man (eg. Lassafever v i r u s , Marburg virus, Zaire hemorrhagic fever virus, e t c . ) . It is d i f f i -c u l t t o imagine more hazardous self-propagating b i o l o g i c a l agents than suchviruses, some of which lead to nearly 100% mortality i n infected individuals.P4 containment requires a s p e c i a l l y b u i l t laboratory with a i r l o c k s and f i l t e r s ,biological safety cabinets, clothing changes f o r personnel, autoclaves withinthe f a c i l i t y , e t c . This l e v e l of containment is required for recombinant DNAexperiments for which there presently is no evidence of hazard, but f o r whichit is perceived that the hazard might be p o t e n t i a l l y serious i f conjecturalf e a r s prove t o be r e a l . There are only four o r f i v e i n s t a l l a t i o n s i n theUnited States where P4 experiments can be c a r r i e d out.3. Experiments associated with a still lesser degree of hypothetical r i s kcan b e conducted i n P3 containment f a c i l i t i e s . These are also s p e c i a l l y const r u c t e d l a b o r a t o r i e s requiring double door entrances, negative air pressure,and s p e c i a l air f i l t r a t i o n devices. F a c i l i t i e s where P3 experiments can beperformed are limited i n number, but they e x i s t a t some u n i v e r s i t i e s .4. Experiments i n which the hazard is problematical and is considered unlikelyto be serious even i f it occurs still require laboratory procedures (P2 containmentl e v e l ) t h a t have f o r years been considered s u f f i c i e n t for research withpathogenic b a c t e r i a such as Salmonella typhosa, Clostridiumbotulinum, andCholera vibrio. The f e d e r a l guidelines require t h a t P2 f a c i l i t i e s be usedf o r work with b a c t e r i a carrying inter-species recombinant DNA molecules t h a thave shown no evidence of being hazardous -- and even f o r some recombinant DNAexperiments i n which there is s u b s t a n t i a l evidence of lack of hazard.5. The P1 (lowest) l e v e l of containment can be used only for recombinant DNAmolecules t h a t p o t e n t i a l l y can be made by ordinary b i o l o g i c a l recombinationprocedures i n b a c t e r i a , Even t h i s lowest l e v e l ofcontainment requires decontamination of work surfaces daily and following s p i l l sof biological materials, decontamination of l i q u i d and s o l i d waste leaving thelaboratory, use of mechanical p i p e t t i n g devices o r cotton plug p i p e t t e s , anda pest control program i n the laboratory.In other areas of actual or p o t e n t i a l biological hazard, physical containmentis 21 t h a t microbiologists have had t o reply upon; i f the Lassa fever v i r u s weret o be disseminated from a P4 f a c i l i t y , - t h e r e would be no f u r t h e r b a r r i e r t oprevent the propagation of t h i s virus which is known to be deadly and f o rwhich no s p e c i f i c therapy e x i s t s . However, even though the hazards ofrecombinant DNA molecules are conjectural, the NIH guidelines have added anadditional l e v e l of safety for workers and t h e public: t h i s is a system of biolo g i c a l containment that is designed to reduce by many orders of magnitude thechance of propagation of microorganisms used as hosts for recombinant DNAmolecules.An i n e v i t a b l e consequence of these containment procedures is t h a t they havemade it d i f f i c u l t f o r the public to appreciate that most of the hazards underdiscussion are e n t i r e l y conjectural. Because i n the p a s t , governmental agencieshave often been slow t o respond to c l e a r and d e f i n i t e dangers i n other areasof technology, it has been inconceivable to s c i e n t i s t s working i n other f i e l d sand t o the pbulic at large t h a t an extensive and costly f e d e r a l machinery wouldhave been established t o provide protection i n this area of research unless severehazards were known to exist. The f a c t t h a t recombinant DNA research hasprompted i n t e r n a t i o n a l meetings, extensive coverage i n the news media, andintervention at the federal l e v e l has been perceived by thepublic as prima facie evidence t h a t t h i s research "must be more dangerous thana l l the rest". The s c i e n t i f i c community's response t h ebeen to e s t a b l i s h - i n c r e a s i n g l y elaborate procedu,res to police i t s e l f -- but thesevery a c t s of s c i e n t i f i c caution and r e s p o n s i b i l i t y have only served to perpetuateand strengthen the general belief that the hazards under discussion must beclear-cut and imminent i n order for such steps to be necessary.It is worth pointing out that despite the scenarios t h a t predict imminentd i s a s t e r from recombinant DNA experiments, the f a c t remains t h a t i n the pastthree and a half years, many b i l l i o n s of bacteria containing a wide v a r i e t y ofrecombinant DNA molecules have been grown and propagated i n the United Statesand abroad, incorporating DNA from viruses, protozoa, i n s e c t s , sea urchins,frogs, yeast, mammals, and unrelated b a c t e r i a l species i n t o E. c o l i . The majorityof these experiments were not subject t o the s t r i n g e n t containment proceduresspecified in the current federal guidelines. However, i n no instance has anyof these recombinants been found to confer a novel hazardous property upon itsh o s t g . c o l i , and i n no instance has any adverse consequence been observed.Despite the absence of detectable hazards i n experiments done thusv a l i d l yf a r , it w i l l always be possible tobargue t h a t recombinant DNA molecules t h a tseem s a f e today may prove hazardous tomorrow: one can no more prove the safetyof a p a r t i c u l a r genetic combination under a l l imaginable circumstances thanone can prove f o r c e r t a i n t h a t the swine f l u vaccine presently being used doesnot contain an unknown and undetected substance t h a t leads to death i n c e r t a i nindividuals, or t h a t an undetected substance capable of producing future canceris present i n currently administered batches of measles vaccine. Nomatter what evidence is collected to document the safety of a new therapeuticagent, a vaccine; a process, or a p a r t i c u l a r kind of recombinant DNA molecule,one can always conjure up the p o s s i b i l i t y of future hazards which it w i l l notbe possible to disprove. When one deals with conjecture, the number of possiblehazards is unlimited; the experiments that can be done to e s t a b l i s hthe absence of hazard are f i n i t e i n number.Those who argue t h a t we should not use recombinant ONA techniques u n t i l o r unlesswe a r e "absolutely certain'' t h a t there is zero r i s k f a i l to recognize t h a t noone w i l l ever be able to guarantee t o t a l freedom from r i s k i nany s i g n i f i c a n t human a c t i v i t y . A l l that we can reasonably expect is a mechanismf o r dealing responsibly with hazards t h a t are'known t o e x i s t o r which appearl i k e l y on the basis of inforkation t h a t is known. Beyond t h i s , we can and shouldexercise caution i n any a c t i v i t y t h a t c a r r i e s us i n t o previously unchartedt e r r i t o r y , whether it is recombinant DNA research, creation of a new drug o r vaccine,or bringing a space ship back to earth from the moon. J u s t as one canspeculate about possible d i s a s t e r s as an outcome of recombinant DNA experiments,one can conjure up s i m i l a r l y frightening scenarios as possible outcomes ofv i r t u a l l y all of mankind's s c i e n t i f i c and i n t e l l e c t u a l endeavors.Today, as in the p a s t , there are those who would l i k e t o think t h a t thereis freedom from r i s k i n , t h e s t a t u s quo. However, humanity continues to be. buffeted by ancient and new diseases, and by 'malnutrition and p o l l u t i o n ;recombinant DNA techniques o f f e r a reasonable-expectation for a p a r t i a lsolution to some of these problems. Thus, we must ask whether we can affordt o allow preoccupation with and conjecture about hazards t h a t are not knownt o e x i s t , l i m i t our a b i l i t y to deal with hazards t h a t -do e x i s t ? We mustask whether there is any r a t i o n a l basis for predicting the d i r e consequencesof recombinant DNA research poctrayed i n the scenarios proposed by some. Wemust then examine the "benefit" side of the ledger and weigh the already- realized b e n e f i t s and the reasonable expectation of additional b e n e f i t s ,. against the vague f e a r of the unknown t h a t has i n my opinion been the focalpoint of t h i s controversy.AddendumDuring recent months, a number of hypothetical scenarios of disastrousoutcomes of "recombinant DNA research" have been w i d e l y , c i r c u l a t e d by opponentsof such research. The following scenarios, although hypothetical, are basedon a c t u a l events,, and are provided simply to i l l u s t r a t e t h a t conjecture canbe applied as e a s i l y to support the need f o r continued research with recombinantDNA techniques, as to support a proposal t o ban such research.Scenario IFact: In l a t e 1976, a previously unknown viral disease characterized by highfevers, severe bleeding, and death occurring i n more than 90% of i n f e c t e dindividuals appeared i n Central Africa. The disease was caused by a virusthought to be r e l a t e d to the "green monkey" v i r u s t h a t appeared i n Europe i n1967 and k i l l e d persons a t West ~ermany's Marburg University who had handledgreen monekys i n Africa. During a 6 week epidemic, t h e hemorrhagic fever diseaseclaimed more than 300 victims i n Zaire and the Sudan before its spread wasd r a s t i c a l l y cut i n late November by the use of s t r i n g e n t i s o l a t i o n procedures.Dr. H. T. Mahler, the Director-General of t h e World Health Organization, whilerecognizing t h a t no s p e c i f i c therapy f o r the disease. e x i s t s and doctors do notknow how the i n f e c t i o n spreads, nevertheless did not believe it was "a very imminent... danger to the rest of humanity". However, Dr. Mahler warned, i n f e c t i o n canspread e a s i l y "in. the kind of world i n which we l i v e , with highways criss-cro ssing AfricaScenario: In September 1983, a deadly disease resembling the hemorrhagic feveroutbreak of 1976 appeared i n East Africa. As i n the case of t h e e a r l i e r Zaire-Sudan epidemic, mortality was nearly 100% i n victims of the disease. I n f e c t i o nspread quickly to medical and nursing personnel from two p a t i e n t s brought to asmall v i l l a g e h o s p i t a l , and then t o a d d i t i o n a l individuals within a 150 squarem i l e area. Despite attempts of the government to cordon off t h e involvedregion, i n f e c t i v e f o c i appeared i n other p a r t s of t h e country and i n a matterof s i x weeks more than 140,000 pe'rsons had d i e d ' from the disease.I n f e c t i o n was spread to Europe by a passenger on one of the l a s t planeloadsof passengers permitted t o leave Africa. In order t o avoid s i m i l a rimportation of the dread disease t o t h e United S t a t e s , the North Americancontinent was sealed off from physical communication with the rest of the worldand the.U.S. Navy and Air Force were i n s t r u c t e d t o use whatever measures a r enecessary to prevent landing of foreign boats or planes on U.S. shores.S c i e n t i s t s .. on several continents working i n t i g h t l y sealedl a b o r a t o r i e s sought desperately but unsuccessfully t o prepare amounts of the v i r u ss u f f i c i e n t t o attempt vaccine production. As a l a s t r e s o r t , i n v e s t i g a t o r s fromseveral American u n i v e r s i t i e s who had been called t o the Public Health ServiceCommunicable Disease Center i n Atlanta were granted permission to begin workingagain with recombinant DNA techniques t h a t had been outlawed by Congress i nmid-1978; t h e i r goal was to construct a b a c t e r i a l s t r a i n able' t o synthesizethe immunologically reactive protein coat of the hemorrhagic fever v i r u s , andto use these b a c t e r i a f o r the production of a vaccine against the deadly disease.Scenario I1Fact: In the F a l l of 1976, the Royal Swedish Academy of Sciences, a t a NobelSymposium on the subject of nitrogen balance, warned about the danger of progressivedepletion of the ozone content of the atmosphere as a r e s u l t of tooheavy use of nitrogen f e r t i l i z e r s . In order t o make nitrogen a v a i l a b l e forgrowing p l a n t s , f e r t i l i z e r p u t s ~ n n a t ~ r a l l ayr g e amounts of f i x e d n i t r o g e n i n t othe. s o i l ; the Academy indicated t h a t this,condition favors d e n i t r o f i c a t i o n , whichreleases n i t r o u s oxide i n t o the air and leads t o the consequent destruction ofozone. The Academy also warned t h a t large amounts of nitrogen f e r t i l i z e ri n h i b i t the n a t u r a l processes of nitrogen f i x a t i o n carried out by legumes suchas peas and beans. When nitrogen f e r t i l i z e r s a r e used to increase the producti v i t y of an open a g r i c u l t u r a l system, the Academy s a i d , the average y i e l d peracre usually becomes progressively smaller and the e f f e c t on the environmentprogressively g r e a t e r as the rate of application increases.The Academy also warned t h a t nitrogenous pollutants i n the air may reactwith amines found i n the body to form nitrosamines, which have been shown tocause cancer i n most organs of experimental animals, and to also be a causeof genetic a l t e r a t i o n s .Scenario: By 1986, the use of f e r t i l i z e r s had destroyed atmospheric ozone to thepoint where t h e World Health Organization had declared an i n t e r n a t i o n a l c r i s i s .Crop yields had become increasingly smaller i n response to f e r t i l i z e r applied.Y e t , increasing use of f e r t i l i z e r was required i n order t o attempt t o feedthe hungry of the world, and an i n t e r n a t i o n a l plea by some nations to ban theuse of nitrogen f e r t i l i z e r s was unsuccessful. An alarming.increase i n ther a t e Of cancer and f e t a l abnormalities was evident i n those countries thatwere the heaviest f e r t i l i z e r users.Although recombinant DNA experimentation had been banned f o r almost tenyears i n many countries, s c i e n t i s t s i n an eastern European nation t h a t hadcontinued t o support such i n v e s t i g a t i o n s announced t h e i r success i n developinga recombinant DNA technique f o r the introduction of nitrogen fixing genesd i r e c t l y i n t o wheat and corn plants, thus eliminating the need f o r the use off e r t i l i z e r i n the growth of these important food crops. Because of the vastp o l i t i c a l and economic implications of t h i s discovery,the United StatesCongress began support of a crash e f f o r t i n recombinant DNA research i n anattempt to develop experimental e x p e r t i s e and fundamental knowledge i n t h i sare a.Scenario I11Fact: In February 1976, the General E l e c t r i c Company was found t o be i n violationof New York S t a t e ' s water policy standards because of its discharge ofpolychlorinated b-iphenyls i n t o t h e Hudson River. The danger was brought t opublic a t t e n t i o n i n l a t e 1975 when high levels. of the extremely s t a b l e chemical- were found i n f i s h taken from the Hudson River, and- t h i s discovery eventually. led t o closing of the r i v e r to most kinds of commercial f i s h i n g . PCB was shownto be a cause of a v a r i e t y of i l l n e s s e s among GE workers, and to producecancer i n laboratory animals; i n Japan, more than 1,000 people were s t r i c k e nwith l i v e r disease and skin problems as a r e s u l t of PCB's t h a t had leaked i n t ocooking o i l . General E l e c t r i c admitted having dumped PCB's i n t o the Hudson.River f o r more than 25 years, and it is believed t h a t by 1976, t h e r i v e r containedabout 500,000 pounds of the toxic chemical. While Congress movedtowards a complete ban on PCB's, s c i e n t i s t s sought a mechanism f o r g e t t i n g r i dof the t o x i c chemical t h a t contaminated t h e Hudson River.,Scenario: The enzymes necessary to carry out p a r t of the degradation of polychlorinatedbiphenols were discovered i n l a t e 1977 to be present i n separates t r a i n s of Pseudomonas putida. Because of a nationwide ban on any researchinvolvingthe joining of genes i n a t e s t tube, s c i e n t i s t s were required to attempt the .use o f -i n -viv o recombinat ion procedures f o r t h e c o n s t r u c t i o n o f a Pseudomonasorganism able to r i d the r i v e r of the PCB p o l l u t a n t s . Microbiological organizati o n s t r i e d i n vain t o convince the New York S t a t e Attorney General's o f f i c ethdt although the needed gene combination eventually might be made by means of theearl ier "trial and e r r o r " -i n -vivo g e n e t i c , recombinat ion procedures , t h e use 'ofrecombinant DNA techniques could provide a -f a s t e r , s a f e r , and more precise methodof achieving the same goal.i .$>i?s< \ STINFORD UXIVERSITY MEDICAL CENTER1 / I 'L- <: STANFORD, CALIFORKIA 94305L--April 6, 1977Hewlett Lee, M.D.Chairman, Committee on Environmental HealthCalifornia Fled i c a l AssociationPalo Alto Medical C l i n i cPalo Alto, CA 94301Dear Huey:I am writing i n connection with Assembly B i l l #757 (copy enclosed) whichhas beenintroducedintothe California Legislature. Hearings on the b i l l w i l lproceed during the next three weeks, and it is my understanding t h a t the b i l lw i l l be reported t o the Assembly by the Committee on Health on April 25. I ' v ebeen advised by sources c l o s e t o the l e g i s l a t u r e t h a t t h i s b i l l , or a modificati o n of it, has an excellent chance of being adopted by t h e S t a t e Assenbly. Ibelieve t h a t passage of the b i l l would have consequences of a most serious naturefor medical research and c l i n i c a l care i n California, and I am writing t o askthe Committee on Environmental Health and the California Medical Associationto oppose the b i l l .So f a r as the s p e c i f i c wording is concerned, the current d e f i n i t i o n of"hazardous biological research" i n AB 757 would cover c l i n i c a l s t u d i e s involvingitems such as throat swabs, sputum c u l t u r e s (TB and other microbes), a d n i n i s t r a -t i o n and t e s t i n g of v i r a l vaccines, etc., -- since all of these things involveI Iorganisms or infectious agents that are capable or can be rendered capable ofcausing wide-spread serious harm, d i r e c t l y or i n d i r e c t l y , t o the health of as u b s t a n t i a l population of humans or t o the n a t u r a l environment". The B i l l wouldalso cover the creation of hybrid plants, ordinary genetic recombination t h a ttakes place n a t u r a l l y in l i v i n g c e l l s , and presumably human procreation -- sincet h i s also involves "organisms ...which are capable, or can be rendered capable,of causing wide-spread serious harm, etc.". Moreover, since there is no evidencet h a t the b a c t e r i a l organisms constructed i n most types of recombinant DNA researchare i n f a c t capable or can be rendered capable of causing wide-spread harm, itis not c l e a r t h a t t h i s research would be covered.The d e f i n i t i o n for recombinant DNA research contained i n the b i l l has beenl i f t e d from the NIH guidelines. However, the d e f i n i t i o n i n the guidelines isabout to be changed; the current d e f i n i t i o n involves regulation of the joiningof two genes derived from the same organism i f such joining is done i n a c e l l -f r e e system -- but not when the joining of the same two genes is done within c e l l s .Since the end products are i d e n t i c a l i n both instances, the current d e f i n i t i o ndoesn't make sense -- and t h i s has led t o a number of administrative problems.Similarly, the d e f i n i t i o n i n AB 757 would cover experiments which everyone agreeswould have no opportunity t o produce novel biotypes or "new forms of l i f e t 1 .Hewlett Lee, M.D. Page twoApril 6 , 1977The b i l l also has a number of other very serious problems. It does n o t .specify the c r i t e r i a to be used f o r determining whether or not the researchbeing regulated is being conducted i n compliance with the law, and it makesv i o l a t i o n of these unspecified c r i t e r i a a felony offense. When it was pointedout to the State Commissioner of Health t h a t the proposed l e g i s l a t i o n wouldhave broad implications for a l l kinds of biological research, he was quoted inthe press a s s t a t i n g t h a t the l e g i s l a t i o n would need t o be modified to excludethose organisms t h a t cause common infectious diseases. Apparently, the Commissionerwould e l i m i r r ~ c o n t r o l over those organisms t h a t a r e known t o be hazardous,and would cover only those organisms for which no evidence of hazard e x i s t s .A t the time Assembly B i l l 757 was proposed, no f e d e r a l l e g i s l a t i o n was inprogress. However, there a r e now several b i l l s pending before the US Senateand House of Representatives, and enactment of one of these is expected withineight weeks with the l e g i s l a t i o n t o go i n t o e f f e c t by July 1. While c e r t a i nmembers of the California Assembly may find it p o l i t i c a l l y advantageous to pursueseparate s t a t e l e g i s l a t i o n , it seems reasonable to ask whether there ares p e c i f i c needs for the protection of California c i t i z e n s t h a t a r e not met by theFederal l e g i s l a t i o n . If so, what a r e they? What is the documentation f o r theseneeds? To what extent would state l e g i s l a t i o n duplicate what is being done a tthe f e d e r a l level?Even i f the b i l l were a good one (and I believe t h a t it is not), it seemsreasonable to ask whether it makes any sense t o have standards t h a t vary amongd i f f e r e n t s t a t e s and communities. Even strong proponents of stringent regulations,such as Sinsheirner, a r e opposed t o standards t h a t vary from s t a t e tos t a t e and from community t o community. Unlike automobile emission controls andc e r t a i n other areas of regulation where it makes sense t o modify standardsaccording to local conditions, it does not appear t o be reasonable i n t h i s area;microbes do not respect s t a t e boundaries, and i n t e r s t a t e t r a v e l is a f a c t ofl i f e .Even a t present there are estimated t o be about 60 separate l a b o r a t o r i e s inCalifornia involving.recombinant DNA research, alone; the proposed b i l l coversmany other areas of research also. If the b i l l were passed, how many inspectorswould be needed to carry out the functions required? How often would they v i s i tthe laboratories? What equipment and f a c i l i t i e s would they need to monitor theresearch? Where would they be housed? What l e v e l of t r a i n i n g would they require,and who would t r a i n them? What standards of e x p e r t i s e for inspectors would beestablished? Who would h i r e and supervise these inspectors, and who would assurethe competence of the bureaucracy t h a t would be established to monitor t h i s veryspecialized research? What advantage would a l l of t h i s costly statewide apparatusprovide beyond what is provided i n the currently proposed f e d e r a l l e g i s l a t i o n ?The proposed f e d e r a l l e g i s l a t i o n has a b u i l t - i n mechanism f o r a l t e r i n gstandards as new s c i e n t i f i c information about hazards, or about the absence ofhazards, is acquired. This involves a national commission in which the majorityof members a r e non-scientists, plus a consultant group of non-scientists and distinguisheds c i e n t i s t s from various areas of research. The technical input at ahigh l e v e l is provided by the NIH, and the NIH budget provides f o r experimentalv e r i f i c a t i o n of hazard or lack of hazard i n p a r t i c u l a r kinds of experiments. WhatHewlett Lee, M.D.April 6 , 1977 page threes o r t of mechanism would there be t o assure technical competence a t the s t a t e l e v e l ,and how would it operate? Would the s t a t e use f e d e r a l l y supplied information, andi f so, would s t a t e decisions using Federal information be any'sounder than Federaldecisions using the same information.At the November 18 meeting of the Committee of Environmental Health, I d i s -cussed my views on the recombinant DNA controversy. The statement I prepared att h a t time led me t o write the enclosed a r t i c l e , "Recombinant DNA: Fact andFiction", which was published i n the February 18 issue of Science, and w i l l bereprinted i n the )fay issue of the Western Journal of Medicine, As noted i n thea r t i c l e , despite the conjectural nature of the hazards involved i n a l l but a fewtypes of recombinant DNA research, most experiments w i l l be permitted only underphysical containment conditions at l e a s t as s t r i c t as those used for work withbacteria t h a t cause typhoid fever and Asiatic cholera. Other experiments requirecontainment f a c i l i t i e s of the type designated f o r the most dangerous n a t u r a lviruses known (eg., Lassa fever, Marburg v i r u s , e t c . ) . Such conditions arerequired for recombinant DNA experiments i n which the evidence of danger is nonex i s t e n t , and the p o s s i b i l i t y of hazard is remote. Moreover, a system of biologi c a l containment is designed t o reduce even f u r t h e r the chance of propagationof recombinant DNA molecules outside of the laboratory. . - . . . . . . . . . .- . . . . . . . . . . . . .I believe t h a t S t a t e l e g i s l a t i o n would be a serious mistake unless thel e g i s l a t u r e can i d e n t i f y s p e c i f i c areas where it f e e l s t h a t Federal l e g i s l a t i o nis inadequate to protect the c i t i z e n s of C a l i f o r n i a , unless it can show t h a tCalifornia l e g i s l a t i o n would increase the safety of the c i t i z e n s of t h i s s t a t e ,and unless it can j u s t i f y the bureaucracy and cost t h a t would be involved i nestablishing and maintaining a state-wide regulatory system.It is worthwhile pointing out t h a t the containment procedures specifiedi n the NIH guidelines, and i n the proposed Federal l e g i s l a t i o n , have made itd i f f i c u l t for the public t o appreciate t h a t most of the hazards under discussiona r e conjectural. Many b i l l i o n s of b a c t e r i a containing a wide v a r i e t y of recombinantDNA molecules have been propagated i n the United States and abroad withoutknown hazard. In c o n t r a s t , work is continuing throughout California and throughoutthe world with infectious disease organisms and with plant pathogens t h a ta r e known t o be capable of causing serious harm. In addition t o the knownhazards presented by such organisms, there a r e a l s o unknown r i s k s associated withorganisms t h a t are known t o cause disease. Yet, as noted above, the S t a t eCommissioner of Health proposes to s e t up a bureaucracy f o r controlling organismsf o r which no evidence of hazard e x i s t s .I'm sorry t h a t it has been necessary t o write such a lengthy l e t t e r , but Ithought it important t o put before you the various issues I am concerned abouti n connection with the proposed s t a t e l e g i s l a t i o n . The b i l l , in its current form,would be a d i s a s t e r . Even in some other form, its p r i n c i p a l value would seem tobe to bolster the p o l i t i c a l careers of some of its proponents --- and the cost ofe s t a b l i s h i n g , t r a i n i n g , and maintaining a huge state-wide bureaucracy t o duplicateHewlett Lee, M.D.April 6, 1977 page f o u rwhat w i l l be done Federally, would be enormous. There is not a great deal oftime to act to defeat t h i s b i l l . I urgently ask your support and the supportof the Committee t o accomplish t h i s .I'd be happy t o discuss t h i s with you i n person or by telephone, i f youwish. With best wishes,Stanley N. Cohen, M.D.Professor of MedicineSNC:db/nmEnclosuresAMENDED IN ASSEMBLY APRIL 19,1977\CALIFORNIA LEGISLATURE-1971-78 REGULAR SESSIONASSEMBLY BILL No. 757Introduced by the Committee on Health, (AssemblymanKeene, Chairman, Assemblymen Agnos, Mangers,Rosenthal, Torres, 4 %eke+ Tucker, Bates, andLantemnan), and the Committee on Resources, Lgnd Useand Energy (Assemblyman Calvo, Chairman,Assemblymen Ellis, Goggin, Hayden, Kapiloff, Wornum,4 Wray, and Bates)March 3, 1977REFERRED TO COMMmE ON HEALTHAn act to add Chapter 10 (commencirig with Section 1780)to Division 2 of the Health and Safety Code, relating tobiological research, and making an appropriation therefor.LEGISLATIVE COUNSEL'S DIGESTAB 757, as amended, Keene (Health). SitAgkd RecombinantDNA research: California 43Megkd RecombinantDNA Research Safety Commission.There is no existing state law governing kaeat$ett4 biekg3eel recombinant DNA research. National Institutes of Healthrkombinant DNA research guidelines apply to l°idrecombinant DNA research in some instances.This bill would enact the California SitAgkdRecombinantDNA Research Safety Act. The bill would create the California3iehgkd Recombinant DNA Research Safety Commissionas an independent commission in state government. Thecommission would be composed of H11members, with 4-47April 25, 1977STATDENT OF STANLEY N. COHEN, M.D.PREPARED FOR THE COFlMITTEE ON HEALTHF,CALIFORNIA STATE ASSEMBLY,I am Stanley Cohen, a physician and Professor of Medicine a t StanfordUniversity -- and a mol.ecular geneticist who has played a role in the developmentof methods for recombinant DNA research,During the past several years we have witnessed an extraordinary sequence.of events involving the interaction of s c i e n t i s t s , the news media, and the communitya t large, It began when biologists learned to duplicate in a t e s t tube the geneticrecombination process that normally is carried on by all living cells. As a r e s u l t ,it became possible t o manipulate hereditary material (DNA), with much greater preci s i o n than had been done previously i n such a c t i v i t i e s as plant and a ~ ~ i mbarle eding,and to combine in a t e s t tube segments of DNA taken from different cells.. Shortly a f t e r the discovery of these techniques, I joined with a group ofs c i e n t i f i c colleagues in calling public attention to the possibility that some genecombinations that could be made using the new techniques might prove to be hazardous.Although there wssno s c i e n t i f i c basis f o r anticipating a hazard, because of thenewness and the r e l a t i v e simplicity of recombinant DNA methods, it seemed reasonableand appropriate to proceed with caution. Further information was needed to enableus to assess more f u l l y the implications of the research, and to determine whetherthere was in fact any risk. In other areas of s c i e n t i f i c and technological development,s c i e n t i s t s and government often had not proceeded as cautiously as they mighthave, only t o f i n d a t ' a later time t.h a t g r e a t e r caut ion had been warranted, Herewas an opportunity in t h i s new research area, we thought, to exercise care a t theonset; i f in the l i g h t of additional experience it subsequently turned out thatour concerns had been exaggerated, then the precautions could be relaxed.Our action was voluntary, and was taken a t the i n i t i a t i v e of s c i e n t i s t sand in the absence of l e g i s l a t i o n or government rulings. Unfortunately, our unprecedentedattempt to guard against hazards t h a t were not known to exist was so novelt h a t it was widely misinterpreted as implying t h a t we thought danger was likely.It has been inconceivable to s c i e n t i s t s working in other f i e l d s and to the publicat large that we would have taken such action, or t h a t an extensive and costlyFederal response to our i n i t i a t i v e would have occured, unless severe hazardswere known.The names of the s c i e n t i s t s who were the leaders in raising the recombinantDNA i,ssue to public consciousness are known to members of t h i s Committee, and someof them have appeared before you a t previous Hearings. However, these s c i e n t i s t shave come here not as proponents of s t r i c t e r controls, but as opponents to the legisla t i o n proposed by the Committee. S c i e n t i s t s such.as Maxine Singer, Paul Berg,Herbert Boyer, James Watson, Ronald Davis, David Hogness, Roy Curtiss, NortonZinder and myself -people who w e r e among the f i r s t to express concerns about thep o s s i b i l i t y that some experiments carried out using these techniques might be hazard-.ous have come to believe t h a t our i n i t i a l concerns were in f a c t greatly exaggerated.Why has t h i s change occurred? Is it because we s c i e n t i s t s are l e s s interested nowi n safety of the public and in protection of the environment than we were j u s t afew short years ago? Is it l i k e l y t h a t t h i s group of s c i e n t i s t s have abandoned t h e i rearlier principles i n order t o simply s a t i s f y t h e i r "ambition" and "curiosity" ashas been suggested by some c r i t i c s ? Or is there another reason why the s c i e n t i s t swho took the i n i t i a t i v e in being cautious have since become the strongest proponentsof t h e research, and t h e opponents o-f e f f o r t s t o implement inc r e a s ingly t i g h t e r contr o l s ? -- -As s c i e n t i s t s , we deal with data, and data are what have led us t o viewt h e s i t u a t i o n d i f f e r e n t l y from before. At the time t h i s issue was f i r s t raised,recombinant DNA techniques were new. Many other kinds of genetic manipulation,such as the creation of hybrid plants and hybrid animals, and modification of theb a c t e r i a l and fungal s t r a i n s t h a t make the a n t i b i o t i c s we use to t r e a t i n f e c t i o n s ,the bread we e a t , and the beer we drink, have been c a r r i e d out for a long time.There was l i t t l e experience i n gene modification using recombinant DNA techniquesat the time we voiced our i n i t i a l concerns. But while the public f e a r s have beenfanned by p u b l i c i s t s and a few s c i e n t i s t s , the work has proceeded without adverseconsequences i n dozens of l a b o r a t o r i e s i n California and the r e s t of the world.During the past four years almost 200 s c i e n t i f i c i n v e s t i g a t i o n s of recombinant DNAhave been published, and l i t e r a l l y hundreds of b i l l i o n s of E. c o l i bacteria containinga wide v a r i e t y of recombinant DNA molecules have'been grown i n the United Statesand abroad with no harm t o humans or t o the environment. Because the l i f e cycle ofa b a c t e r i a l c e l l l a s t s only 30 minutes, it has been possible t o study many thousandsof generations of organisms containing recombinant DNA molecules. What has beenfound i n these s t u d i e s has persuaded us t h a t the conjectural f e a r s t h a t led t o most,i f not all, of our e a r l i e r concerns a r e without basis i n f a c t .Dr. Roy Curtiss is a distinguished microbiologist who because of h i s deeplyheld concerns has spent much of the past three years designing and developing f a i l -s a f e b a c t e r i a l s t r a i n s f o r genetic experiments. In the course of these s t u d i e s ,Dr. Curtiss has accumulated much information t h a t has modified h i s own thinking,and which has contributed t o a l t e r i n g the thinking of the r e s t of the s c i e n t i f i ccommunity. Dr. Curtiss, who from the beginning has been one of the most cautiouss c i e n t i s t s i n t h i s controversy, has recently written a 13-page l e t t e r t o Dr. Donaldkedrickson, Director of the National I n s t i t u t e s of Health. H i s l e t t e r s t a t e s :"In view of a l l the accumulating information...I have gradually come t o the r e a l i - -zation t h a t the introduction of foreign DNA sequences i n t o EK1 and EK2 host-vectors- -o f f e r s no danger whatsoever t o any human being with the exception already mentionedof an extremely careless worker who might under a unique s i t u a t i o n cause harm t ohim or herself". A similar conclusion was reached regarding the absence of hazardt o the biosphere. "The a r r i v a l of t h i s conclusion has been somewhat painful," Dr.Curtiss s t a t e s , "and with reluctance, since it is contrary to my past ' f e e l i n g s 'about the biohazards of recombinant DNA research."Along with the r e s t of us, Dr. Curtiss i n i t i a l l y feared the spread of bacteriacontaining recombinant DNA and the possible conversion of harmless to harmful bacte r i a . However, much experience and data have shown us t h a t the introduction ofrecombinant DNA molecules i n t o already weakened laboratory s t r a i n s of -E. c o l iweakens them even f u r t h e r , so that they a r e unable t o compete successfully i nnature and a r e a t a disadvantage outside the s p e c i a l protective conditions of thelaboratory. Moreover, extensive s t u d i e s have shown that even genes known to codef o r disease t r a i t s f a i l t o make disease-producing bugs out of laboratory s t r a i n s ofE. c o l i .Dr . C u r t i s ~g oes on t o s ay t h a t he has "become i n c r e a s i n g l y d i s t r e s s e dby degeneration of the debate. Opinions have often been s t a t e d as f a c t u a l c e r t a i n t y ,statements of ' f a c t ' have often been put f o r t h t h a t a r e i n c o n f l i c t with publisheddata and there has often been an unwillingness t o adhere t o the principles ofs c i e n t i f i c o b j e c t i v i t y . I have never heard or read any f a c t u a l information i n thedeba t e t h a t would c o n t r a d i c t t h e conclus ion about t h e s a f e t y of t h e -E. -c o l i IC12host-vector system that I have j u s t reached. It is thus my considered belief t h a twe a r e about t o embark on excessive regulation of an h p o r t a n t area of biomedicalresearch based almost solely on f e a r , ignorance, and misinformation".Mr. Chairman, what is proposed by t h i s Committee is akin t o establishingl e g i s l a t i v e r e s t r i c t i o n s t o prevent the continued use of a drug or vaccine t h a t hasalready been used f o r several years without any adverse consequences, and whichaccording to a l l experimental evidence is not associated with any r i s k whatsoever_*_- - --- simply because there a r e some who have unsupportable anxiety t h a t a still unknownhazard might possibly occur some time hence.There a r e a few s c i e n t i s t s who continue, f o r almost mystical reasons, to f e a rhazards even where experience has told us there are none. However, the views ofthe r e s t of the s c i e n t i f i c community have s h i f t e d as data have gradually becomea v a i l a b l e to allow us t o replace speculative concern with f a c t . Careful exaninati o n of the issues by the public has led them t o a p a r a l l e l change i n perspective.The Cambridge, Massachusetts Citizens Committee has concluded t h a t "many of thef e a r s held by the c i t i z e n r y r e s u l t from a lack of understanding about the nature ofthe research and the manner i n which it is conducted". When the f a c t s were examinedc r i t i c a l l y by the Cambridge committee, as Time Magazine recently reported, t h e .I tc i t i z e n s patiently ignored p o l i t i c a l demagoguery, perceived the f a l s e notes i n thevoices of doom, mastered the complex issues, and then passed t h e i r votes for continuation-- with reasonable r e s t r a i n t s -- of f r e e s c i e n t i f i c inquiryv. The unanimousposition of the Cambridge committee, which r e s u l t e d from seven months of intensivestudy, indicates t h a t when non-scientists spend the time to examine carefully theissues, they reach the same i n e v i t a b l e conclusion as most s c i e n t i s t s -- namely t h a tpredictions of d i s a s t e r a s a r e s u l t of continuation of recombinant DNA research a r ewithout any reasonable basis i n f a c t .Unfortunately, the perception of most of the public on t h i s issue has laggedbehind the s c i e n t i f i c data. It is i r o n i c t h a t even as evidence has accumulated tosupport the view t h a t there a r e no novel hazards associated with the research, thef e a r s of the c i t i z e n r y have increased, and most of the public now believes t h a textensive l e g i s l a t i v e control mechanisms are necessary. Hearings were held ont h i s issue by the U.S. Senate i n April, 1975 and i n the f a l l of 1976, and by theHouse of Representatives l a s t month. When t h i s Comnittee of the California Assemblybegan to investigate the issue, no Federal l e g i s l a t i o n was in progress. However,at t h i s point, the Federal government is about t o provide the public with an "insurancepolicy" in the form of a Federal l a w on recombinant DNA, and more than half adozen b i l l s ' a r e now pending before the U.S. Senate and House of Representatives.Two weeks ago, in revising AB 757, the Assembly Committee on Health d e l e t e d 'the p a r t s of its e a r l i e r b i l l t h a t would have supplemented the proposed Federall e g i s l a t i o n , and has now developed a b i l l t h a t simply duplicates what is being donea t the Federal level. While I r e a l i z e t h a t California has the r i g h t to e s t a b l i s hits own l a w s i n t h i s area, I seriously question the usefulness of doing so.F i r s t of all, microbes do not respect s t a t e boundaries. People enter andleave California every day, and i f there is any r i s k from recombinant DNA work, thec i t i z e n s of California can be no s a f e r than the c i t i z e n s of other s t a t e s regardlessof what standards a r e l e g i s l a t e d i n Sacramento. Even the most vocal proponents ofs t r i n g e n t controls such a s Professor Robert Sinsheimer, have come out strongly f o rFederally administered regulatory procedures, r a t h e r than f o r s t a t e or l o c a l legislation.As someone familiar with t h i s area of research, I can assure the Committeet h a t extensive f a c i l i t i e s and personnel would be needed to carry out any monitoringa c t i v i t i e s established by s t a t e l e g i s l a t i o n . Where would the people and equipmentbe housed? Who would t r a i n the inspectors and where would they come from? Theproposed Federal l e g i s l a t i o n has a built-in mechanism for modifying standards as news c i e n t i f i c information is accumulated. This involves a Federal Commission in whichthe majority of members a r e non-scientists; the Commission would use the NIH a s aresource and repository o'f s c i e n t i f i c data and technical expertise. What s o r t ofmechanism would the S t a t e e s t a b l i s h t o assure technical competence? Would Californiause Federally supplied information, and i f so, would S t a t e decisions using t h i sFederal information be any sounder than Federal decisions using the same information?What, i f anything, would be gained by s e t t i n g up a l i s c e n s i n g , monitoring,and enforcement system i n -California t o duplicate the Federal system?In the absence of any, foreseeable benefit to Californians or any demonstrable- ---need for State l e g i s l a t i o n , the band-wagon nevertheless r o l l s on. W e have beentold that the process can always be halted l a t e r on i f Federal laws prove to besatisfactory. However, past experience suggests that once a mechanism for a regulatoryapparatus is established, it w i l l be d i f f i c u l t to pull back -- even thoughFederal laws w i l l make a Statewide apparatus unnecessary and undesirable. ~ o e s n ' tit seem preferable to determine whether there is a need for a California l e g i s l a t i v esolution before proceeding, rather than to proceed h a s t i l y with the expectationt h a t the time and e f f o r t invested w i l l not have been useful?A reasonable alternative to the present version of AB 757 is the establishmentof a study commission - rather than a regulatory commission -- to evaluatecalmly and responsibly the data that have become available on t h i s subject, to sortout polemic from f a c t , and to analyze the claims made- on both sides to see whatarguments can r e a l l y be substantiated.Time constraints prevented the City Council of Cambridge from carrying outthe kind of in-depth analysis of the issues made by t h a t c i t y ' s c i t i z e n s committee-- and I suggest that similar t i m e constraints have prevented an in-depth analysisby t h i s l e g i s l a t i v e committee - even with s t a f f assistance. Examination of the. h a s t i l y prepared t e x t of the current version of the b i l l seems to support t h i s view.Does California deserve a l e s s deliberate and thoughtful process than Cambridge?Can t h i s committee carry out the in-depth analysis of the Federal l e g i s l a t i o n neededto detem'ine the adequacy of the extensive and complex Federal regulations?Careful study is required to determine whether there are any needs for the protectionof c i t i z e n s of California against biological hazards that are not alreadyprovided for in the Federal law. If so, a study comission could identify theseneeds, whether i n recombinant DNA research per -se or in other areas of biologicalexperimentation and could then recommend appropriate l e g i s l a t i v e action. If currentl a w s and the pending Federal regulations a r e found to be adequate, nothing would begained by establishing a separate regulatory and licensing system in t h i s State.Recombinant DNA research was born i n California, and s c i e n t i s t s in t h i sS t a t e were the leaders i n r a i s i n g t h i s issue to public awareness. This Committeeof the Assembly now has the opportunity t o continue the p a t t e r n of Californialeadership i n t h i s f i e l d , not by following a "me-too" approach f o r duplication ofwhat is being done on the Federal l e v e l , not by rushing ahead with h a s t i l y draftedand even more h a s t i l y modified l e g i s l a t i o n on an issue of such major importance,but r a t h e r by adopting a c r e a t i v e , d e l i b e r a t e , and responsible l e g i s l a t i v e approach.I believe that such an approach involves c a r e f u l study of the issues and of thepending Federal l e g i s l a t i o n , r a t h e r than approval of AB 757.
September 6, 1977Donald Fredriclrson, PI. D.DirectorN a t i o n a l Institutes of HealthWthesda, MD 20814Dear Don: . -I have enclosed a copy of that is now in press inProceedings -of -the National Academy of Sciences. I have taken the unusualstep of senking i t to you prior to p s l i c a t i o n because I believe the findingsMva policy, as w e l l as scientific, importance w i t h regard to theregulatfon of reawmbinant DM. .-"1;The experinrents reported i n the paper demonstrate that:(I) X R X restriction endonuclaase occurrfng within normally growing bacterialcells promotes genetic recombination i n vim at precisely the same sites thatare involved ia i n vitro recombinant DNA experiments using this enzyme, and(2) Free f r a p n t s of eukarpotic DNA can be taken up by E. coli and joined toplasmid JSA rsoleculss within rhe bacteria. The fesulting hybrid eukaryoricpmkaryoticDNA mleeules (which have been made i n t r a c e l l d a r l y by naturallyoccurringbiological processes) can be propagated i n bacterial cells by theplasmid replication system.These eqeriments and others have led us to conclude that an importantbiological function (perhaps the major function) of the so-called "restriction"enzymes may be site-specific recombination of DHA, and e a t iaaktzryotie DNAfragnwints formed biologically by restriction en- cleavage can link toprokaryotic DNA M a t o i n vitro recombinant DNA techniques. Our data providecompelling evidence to support the view that reambinant DNB. molecules constructedi n vitro using the e R I enzm simply represent selected Pnstacesof a process that occurs by natural means. - - ; p r . .,-2. :-QK~%$$~:.-i * a- >:,&-.2-- .I n the pas t I believed that the i n v i t r o joining of di f ferent seg-4 - -,-. .* -+.i+Y>5: zm a t s of DNA at restriction agdonuclease cleavage sites resulted i n the ' ' ,-..*,* 5 , _ - - I.-7 formation of genet ic combinations tha t a u l d nor be made otherwise; f o r this '*%+-' . -reason it seemad important to c d l attention to possible biohazards that might - ,:,',-* be associated with certain kinds of novel gene combinations. However, along . .* w i t h virtually all of the other s c i a n t i s t s who first raised these questions,. 2I have sfnce come to believe that our i n i d a l concerns were greatly overstated..,Some of the important new information that has led to this changed perceptionDonald Fredrickson ,M. D..September 6, 1977Page 2has been smaarized in Roy curtis' recent Letter to yon and in tire Fdmoutbc:. ': :report. The data described in tha enclosed maauscript: at test to the LT... 2 ;..';...~..'...:~...... ..,. .......-..;. . ,..:...:-, ..;. . .naturalness of site-specific genetic recombination mediated by restriction .. -:;f;-.i-.-:endonucleasw and intracellular ligases, and add still another perspective a.' ;. :I.;.:; I-.: +:;: .;. . . ., the controversy, . .-.--..%--:,,:-.+.::-.*....,.....,.: :.,I. -. '.-I . ..., i; ;;.:+ajpg5z..c;-<*.2 i'... .., ,-.>;. .. . .. -5. " *.. I would be happy to answer any specific questions that you m y have‘,:.'^.':;;-.:;:.^^^^:-- .. .. ..-%. -.:-?b - - :...* - . .$y*. >,-. : . . about this work, . .' - .- ,2.'. ..*-:4z.:- " *, 7- -. \.- :7 ,*,+<+, . . a . . . .. ,..,r.>:<,-.&.*v;&*.z -... ... ,..,:.+2*-z&d*2@; .. -2 $;>..a-.,- : < : - . With be s t wish-, :. , . ..Sincerely yours,Stadley N. Cohan, M, D,Professor of Medicine andProfessor of GeneticsSLJC: sehEnclosureFFICf .MEMT?RANDUM, STANFORD UNIVERSITY OFFICE MEMORANDUM STANFOZD UNIVERSITY OFFiCE MEMORANDUM/ -DATE: 10 J u l y 1978TO : Josh Lederberg...FpoM : Stan CohenSUB'ECT: Our discussions about the o ~ i g i n sof the "recombinant DXA technique"Dear Josh:Xicholas Wade's inquiry about "inventorship of the recombinant DNAtechnique" has prcmpted me to p u l l together and s e t down on paper mythoughts about the s c i e n t i f i c contributions i n t h i s area. This l e t t e rprovides you with these views, a s you have requested.Since s c i e n t i f i c knowledge is obviously a continuum, and s i n c eeach discovery is dependent upon others t h a t have gone before, Wade'si n q u i r y about s c i e n t i f i c "inventor ship" when cons idered i n a broad s-ense,r a i s e s a v a r i e t y of philosophical and e t h i c a l i s s u e s . Some of the geniralconcepts involving recombinant DNA depend on the work by Avery,MacLeod and NcCarty, on Watson and Crick, on your own work, and onothers; these advances depended i n turn on the preceding contributionsof others. However, i n a more,narrow sense, the answer to Wade's questionabout inventorship of t h s recombinant DNA technique depends i n p a r t onwhat one'regards a s "the recombinant DNA technique".Most observers consider "recombinant DNA" t o be conceptually equivalentt o gene cloning r a t h e r than gene splicing--although they may not havethought abcut t h i s d i s t i n c t i o n e x p l i c i t l y , and recombinant DNA has beenref erred to popularly a s "gene splicing". The conceptual and experimentalelement s of gene s p l i c i n g -per -s e can be found i n t h e work of Khorana andh i s c o l l a b o r a t o r s , who i n the l a t e 1960's showed t h a t s h o r t segments ofs y n t h e t i c DXX could be spliced together by the a d d i t i o n of overlappingconpleinentary single-strand t a i l s (sumarized i n Agarwal e t a l . , Nature-227, 27, 1970) . The u s e of t e rmina l t r a n s f e r a s e t o add homopolyineric dAand dT t a i l s t o the DXA segments was f i r s t reported by Jensen e t a l . , 1971(Biochem. Biophys. Res. Corn. 43, 389, 1971). In these conceptually sound,but only p a r t i a l l y successful z p e r i m e n t s , s e p a r a t e DNA molecules werelinked together by d4-dT t a i l s to form catenanes; however, Jensen e t a l .did not achievs th.e f i n a l step of l i g a t i o n t h a t was necessary to accoiplishDNA s p l i c i n g . The paper by Jackson, Symons and Berg (PNAS 69, 2904, 1972)which was the f i r s t to report success with the dA-dT methodTf joining,c r e d i t s Lobbzn and Kaiser with i n i t i a l l y making the observation t h a t exonucleaseI11 was needed to acconplish what Jensen e t a l . had f a i l e d to l o :namely, the covalent joining of Dh"l molecules. t h a t have horr,opolymericextensions of dfi and dT a t t h e i r ends. The Lobban andKaiser work waspublished i n mid-1973 (J. Pfol. Siol. -78, 453, 1973).--Femo t o J. Lederberg 2 10 July 1978As Paul Berg has indicated, concern about possible biohazards r e l a t e d- t o the SV40 component of the Xdv-SV40 molecule t h a t Jackson e t a l . had const r u c t e d , led him t o decide not to t r y t o clone the molecule i n E. c o l i .However, there is no report of success i n the cloning of analogous moleculesthat' contain any other fragment of DNA i n s e r t e d at the Xdv site used i n theJackson e t al. experiments. Apparently the reason for t h i s is t h a t the-EcoRI cleavage site i n Xdv is located within the 0 gene (Helling et a l . ,.J. Virol. 14, 1235, 1974; Streek and Hobom, Eur. J. Biochem. 57, 595, 1975;Mukai et a c , Mol. gen. Genet. 146,269, 1976) which is e s s e n x a l f o r r e p l i -cation. Interruption of the continuity of t h i s gene by an i n s e r t e d DNAfragment prevents Xdv from functioning a s a r e p l i c o n .Later i n v e s t i g a t o r s have succeeded i n using EcoRI-cleaved Xdv a s ' acloning vector by constructing molecules t h a t contain Xdv dimers (Xukaiet al., Mol. gen. Genet. 146, 269, 1976) plus the fragment to be cloned.In t h i s case, one of the two copies of Xdv provides an i n t a c t 0 gene andt h e molecule is thus able t o r e p l i c a t e when introduced i n t o b a c t e r i a lc e l l s . However, intermolecular linkage of DNA molecules of the sane speciest o form dimers is prevented when the dA-dT joining method is employed(Jackson et a l . , PNAS -69, 2904, 1972).As I wrote i n S c i e n t i f i c American (July, 1975), I b e l i e v e t h a t thegene cloning technique depends d i r e c t l y on discoveries made i n a numberof d i f f e r e n t l a b o r a t o r i e s i n the l a t e 1960's and e a r l y 1970's. The componenttbat involves the s p l i c i n g together of DNA segments by means of addedcohesj.ve termini t r a c e s its conceptual and experimental o r i g i n s to Khorana'swork, a s noted above, and the joining of s e p a r a t e DNA nolecules by meansof r e s t r i c t i o n endonuclease-generated cohesive termini waS reportedsimultaneously by Nertz and Davis (PNAS 69, 3370, 1972) and by Sgaramella(PNAS 69, 3389, 1972). The discovery t h a t r e s t r i c t i o n endonucleases canrecognize and cleave DWA a t s p e c i f i c nucleotide sequences was made by Kellyand Smith (J. Mol. Biol. 51, 393, 1970) and the f i r s t use of these enzymesf o r r e s t r u c t u r i n g DXA molecules by cleaving them i n t o fragments and joining..Li;:e r e s u l t i n g segments together i~a - d i f f e r e n t a r r a n g a e n t was reportedby Cohen e t a l . (PNAS 70, 3240, 1973).The discovery and p u r i f i c a t i o n of DNA l i g a s e by G e l l e r t (PNAS 57, 148,1967) and others was important i n enabling covalent linkage of sepziEteDNA molecules -i n -v i t r o . However, l i n k a g e of p h y s i c a l l y s e p a r a t e r e s t r i c t i o nendonuclease-generated DNA f ragments can a l s o be accompl ished -i n -vivo bythe DNA l i g a s e (Cohen e t a l . , PNAS 70, 3240, 1973). In f a c t , r e s t r u c t u r i n gof DNA mo l e c u l e s by t h e combined i n ~ a c e l l u l a ra c t i o n s of r e s t r i c t i o n endonucl e a s e and DNA l i g a s e can be done -i n -vivo a s we l l a s -i n -v i t r o (Chang andCohen, PNAS 74. 4811, 1977). Cchesive termini a r e not e s s e n t i a l for thelinkage of DKA segments; the work of Sgaramella e t a l . reported i n 1970--------Memo t o J. Lederberg . 10 July 1978(PNAS 67, 1468, 1970) showed t h a t even blunt-ended DNA fragments can bej o i n e d X g e t h e r by use of the bacteriophage T4 ligase.In addition t o a method f o r s p l i c i n g together d i f f e r e n t DNA segmentsat s p e c i f i c sites, the -- biologically f u n c t i o n a li n v i t r o construction ofDNA molecules (Cohen et a l . , PNAS 70, 3240, 1973) is dependent on the conce p t of u s i r g a v e c t o r t o i n t r o d u c e ~ Ni n~t o a r e c i p i e n t c e l l and t h edevelopment of methods f o r accomplishing introduction o f . t h e vector experimentally.Several d i f f e r e n t systems f o r introducing bacteriophage DNAi n t o E. c o l i were described i n the 1960's. Piandel and Higa (J. Pfol. Biol.53, 159, 1970) f i r s t reported the use of calcium chlorLde to acc&rnplishuptake of bacteriophage DNA i n t o E. c o l i K 1 2 , and the production ofv i a b l e phage p a r t i c l e s ( i . e . , t r a n s f e c t i o n ) . However, these i n v e s t i g a t o r sreported t h a t they were unable to generate b a c t e r i a l transformant clones.Such t r ans forma t ion, and the propagat ion of c l o n e s of -E. -c o l i cont a iningr e p l i c a s of introduced DEAmolecules was first reported by Cohen, Changand Hsu (PNAS -69, 2110, 1972), using plasmids.The recombinant DNA technique a l s o depends on a means of selec?ingfr0m.a l a r g e population of r e c i p i e n t c e l l s those individuals that havereceived e i t h e r chimeric or r e s t r u c t u r e d DNA molecules, and upon thediscovery t h a t foreign DHA can be propagated i n c e l l s using a repliconindigenous t o the recipient (Chang and Cohen, PNAS 71, 1030, 1974;~ o r r o re~t a l . , PXAS 71, 1743, 1974) . Thi s l a s t was n o t a foregoneconclusion--since t h e g e n e t i c and s t r u c t u r a l s t a b i l i t y - of i n v i t r oconstructed DSA molecules and t h e i r capacity f o r biological functionwere not c e r t a i n before the experiments were a c t u a l l y c a r r i e d out. Inf a c t , some DSA chimeras are not s t a b l e or b i o l o g i c a l l y viable--and to t h i sday c e r t a i n DSA combinations cannot be cloned.I appreciate your tzking the t i m e t o t a l k about t h i s matter with me.With b e s t wishes,Sincerely yours,b t PAPPLICATION FOR APPROVAL OF RESEARCf-i PRWECT INVOLVING RECDBINANT DMIIdentification No, oft h i s applications C - / o /Name. and Title of Principal Investigator:Stanley N. Cohen, Professor-. . .Department:Genetics. .Title of Grant: .Telephone No. (415) 497-53157P . -- . .. . . .. . . . . .Gene Expression i n Heterospecffic EnvironmentsResearch Su ort (Agency and Grant NO.): New Renewal 0 tonti.nuatlon ~1Specify to whom.jnstitutional approval should be sent) + Division of Research Grants Central Processing SectionNational I n s t i t u t e s of Health National Science FoundationBethesda, Maryland. '20205 Washington, D.C. 20550 ,A. Description of Project: .-: .1. Description of Experiment: (Indicate whether experiment involves use ofa1ready constructed DNA molecules, organisms already containing recombinantDNAs or whether each of the above is to be constructed).The experiments w i l l involve further work with cDNA sequences for mousedihydrof o l a t e 'reductase . An MU4 covering t h i s workhas been approved previously; the present MUA is a request for reductionof containment level, since the plasqids previously constructed have nowbeen rigorously purified by cloning, and the DHFR cDNA segment has beensequenced. The conditions specified i n footnote 3 of the NIH guidelinesof December 22, 1978 have been met, since the segment cloned consistse n t i r e l y of DHFR cDNA as determined by DNA sequence analysis. Lot~ering. - of containment f o r these very same clones has already been allowed foranother lab at Stanford,The prbposed experiments w i l l involve the.introduction of segments of thesequenced cDNA into E. c o l i plasmids that contain characterized transcriptional - - .and t r a n s l a t i o n a l control signals, and the study of expression of the DH'FRenzyme i n such clones. he DNA i t s e l f ' w i l l be analyzed by r e s t r i c t i o n endonucleasemapping and DNA sequencing procedures employing ge1.electrophoresis.C2. Source of DNA to be Cloned:(Indicate species organ or tissue, chromosomal,extrachrorsosoinal or organel le).AT-3000 mouse c e l l s- -- - - -3. Purity of DPiA to be Cloned (e.9. comznt 01) klnexner exPS-ltnent involvesshotgun cloning of total DNA, prior purification of organelle, puriff ationof CCC DtIA by cesium choride-ethidi um bromide centrifuga-tion CDNA f r o m RNA, etc. ):-Previously cloned and sequenced. .4 . Criteria for Purity of DNA to be Cloned (ifrelevant to the containment1eval proposed): . - . .'.. , . ;. ... . . . . .. -. ... . . . . . - . ,.. ;4 -. . . . : . . ..,..d:-'- '. . . . . . . . . . . . .-- :.:.- ..-. . .. . .. . . - . . . - . . . - . .. ...,: >: +.. :. . . . . . . . . .:. .'. . .. . DNA sequence .hasbeen determined f o r already. cloned DNA. species . , ' . . . . . . . . . . . . . . .I*. . ... . - . . .- . .- . . . - . " . . . . ,. . . . . . . . . . . , . . . .' . . . . - . . ' . ' . .. - . . . . 1. .: . . .- . . . . ,* - .: 5 . \fetector(s): . . . _. .. . . -.pSClOl and pBR322 (EK~approved). - pACYC184 (EK1). .F . . -Also o t h e r E K1 ve c t .o .r s d e r h e d from these plasmid replicons.. ..6. ~ o s t r-(Bnd strain 'if relevant; eeg. recoli, C600, ~1776,& ~ ~ b t j l f s ,'etc.)-. .E. c o l i K12 strains.8. e v e 1s of Physical and Biological Containment:. Levels of Containment recomnehded by HIH Guide1ides, RELEVANT SECTIONS$UST BE CITED.. . . .Section' III-A-l-a (2) ' specifies P2 + EKZ..III-A-3 Reduction of containment l e v e l for. purified DNA .;.:;and III-A-3-b (112 that has been rigorously characterized ( t o P2 + EG)2. Level of Physical Containinent to be Used, identify location (building, .room number, c i t y .and state) and specific procedures used,to providerequired levels of containment:P2 - S-141, S-175, Medical Sciences Buildingalso L-314 a f t e r lab moves in April, 1979. Rooms are a l l i n Stanford, CA,The containment procedures for P2 experimentation as specified in the NIHguidelines of December 22, 1978 w i l l be used.IC. Project Personnel* (1?st a1 1 personnd involved in the conduct of these 'experiments): . .1. Names, ~ i t l e sand Responsibilities; . ...* S. N. Cohan, Principal Investigator, Professor of ~enetics . -A.C.Y. Chang, Life Science Research Assistant and Graduate Student ..2, S t a t e of training of laboratory personnel working Oc project regardinappropriate containment proceduns (For projects requiring conta jnme:tcondi t i o n s h i g h ~ rth an P2 iKl ,.d m r ibe what t r a i fling the personnel havereceived) :Extensive kxperience (5 years) working with recombinant DNA and containment,3. Fami l i a r i ty of professional personnel t ~t ih the fJIH Guide?ines:Have read and understood guidelines '4. Information on Health ~urvei1lance:-Rot applicableD. Additional Com~ents:I agree to co~iplywith the NIH requirements pertaining to shipment andtransfer of recombinant D:SA materials. I am familiar with and agree toabide by the provisions of the current HIH Guidelines and other specificNIH instructions pertaining to the proposed project, The informationabove is accurate and complete.'%dL\Z)Date.. . Principal Investigator -I assure that the Administrative Recombinant DNA Panel has reviewedthe proposed project and the plans for facilitiesproposed or under construction or renova tion , Recombinant DMA experjmentationwill not occur until the conpleted facilities have been reviewed by thePanel and a HUA w i t h certification has been submitted to NIH,The Panel as delegated by the Institution agrees to accept' responsibil jtyfor the training of all laboratory workers involved in the project. ThePanel will monitor throughout the dut-ation of the project the facilities,procedures, and the training and expertise of the personnel involved i n thereconbinant D M activity.Date Chai rmanAdministrative Recombinant DNA PanelDate Institutional Official . .Date Institutional Official*. .*Additional pel-for~qances i tes, if appl icable.I agree to comply with the flIH require~entspertaining to shipment andtransfer o f recombinant Dt1.4 materials, I an far~iliar ~ j t hand agree toabide by th= provisions of the current F!IH Guidelit~esand other specificRIH instructions pertaining to the proposed project. The infomationabove i s accurate and complete.. .. - %13, GwDate --: . . .. .- . . - _ . - -'.:- .. . ,. -.::.-... .i---. :. - _ ., . . -..-.. . . . . . _ - ' .. .. . . . . . . - .. .-.. . .- * . . .. . . . . . _... : -. . .... . -. -. . ..... ..- .- . . -' -.... .:.' ' . - . .I certify that the Administrative Recombinant ENA Panel has reviewed on .the proposed project for recombinant DfU! experf-.nents and has found it to be i n co~pliancewith th? NIH Guidelines andother specific NIH instructions pertaining to the proposed project. .The Panel as delegated by the Institution agrees to accept responsibi? ityfor the training of al l laboratory workers involved i n the project, ThePanel will monitor throughout the duration of the project the facilities,procedures, and the t r a i n i n g and expertise of the personnel involved i nthe recombinant DNA activity.. -.- . . . . . . . .Date ChairmanAdnini stra t i v e RecombinzntDDEIA PanelDate Institutional OfficialDate Institutional Official* I*Additional perforlnance sites, if appl icahl e.For scientists (graduate students, post-doctoral fell ow, visiting scfentjsts,etc.) engaged in experiments using reconbinant DNA techniques,1 an familiar with the containment procedures described i n "The NIHfor Research Involving Recombinant DNA Eolecul es" that are appropriate for mywork.. -I agree to abide by the provisions of the llIH ~uidelinesin all experiments 1perfonn a t Stanford University with such molecules. - .i N A M E j .. Print .. .. .. I agree to abide by the provisions of the t4IH Guidelines for RecombinantDNA t4olecules and that the Recombinant DNA Molecules being used willnot be transferred to other investigators or'institr.~tions unless they haveprovided written assurance that their facilities are adequate and their procedureswill be carried out i n accordance w i t h the ClIH Guidelines and otherEIIH instructions, an approved MUA i s on file and a copy of the requesthas been filed \vi t h their committee. Prior to shipment of recombinant DNAmaterials to a foreign country, I shall obtain from the requesting laboratory -a statement t h a t the research involving recombinant D3A molecules w t ' l l be conductedin accordance with the containment standards of the NIH Guide1 ines,. orunder appl icable national guide1 ines.The sending investigator shall'maintain a record of all shipnentsof . , , ' recombinant DNA materials.. -. .N-O--TE-: A) For projects requiring Panel review include this complete MUAsigned by the Principal Investigator with copies as follows:1. 17 copies of pages 1, 2, and 3 and any supportingdocumentation i f 1)cccssar.v.2. For new projects include one copy of the Grant Proposal.8) The )IUA, copies, and grant proposals should be sent to JackSidlow a t 71 Encina Hal 1, Ext, 7-3201..THE HARVEY LECTURES. SERIES 74THE TRANSPLANTATION ANDMANIPULATION OF GENES INMICROORGANISMSXSTANLEY N. COHENDepartments of Genetics and Medicine,Stanford University,Stanford, CaliforniauUNTIL this decade, genetics has been largely a descriptive science:our knowledge of genes and their actions has been derived mostlyfrom observing the consequences of natural biological processes such asmutation and recombination. Certainly, the ability to introduce newgenetic information into bacterial cells by the manipulative processes oftransduction, transformation, or conjugation has advanced knowledgeof the biology of prokaryotic organisms in major ways, and concurrentprogress in biochemistry and molecular biology has enabled the structuraland functional study of the individual genes and gene products ofprokaryotes. However, the complexity of the chromosomes of higherorganisms and the inability to isolate particular segments of these DNAmolecules has until recently precluded detailed molecular analysis ofeukaryotic genes.Development of the concepts and methods of "recombinant DNA"now enables the manipulation of DNA molecules in vitro and the cloningof new genetic combinations in microorganisms. This has permittedthe investigation of prokaryotic genes at a level that was not previouslypossible and has allowed for the first time the analysis of individualeukaryotic genes and study of the organization of genetic information in .higher organisms. The advances that laid the foundations for geneticmanipulation in microorganisms were made in a number of differentlaboratories in the late 1960s and early 1970s. There are four generalrequirements: (a) a replicon (cloning vehicle or vector) able to propa-*Lecture delivered May 17, 1979.174 STANLEY N. COHENgate itself in the recipient organism; (b) a method of joining anotherDNA segment to the cloning vector; (c) a procedure for introducing thecomposite molecule into a biologically functional recipient cell; and (d)a means of selecting those microorganisms that have acquired the hybridDNA species.11. HISTORICABLA CKGROUNANDD THE DEVELOPMENOTF DNACLONINGMETHODSA. Plasmids and Plasmid DNA TransformationThe studies reviewed here grew out of experiments aimed at elucidatingthe molecular nature of a class of genetic elements responsiblefor antibiotic resistance in bacteria. It has been known for some yearsthat many bacterial species contain autonomously replicating extrachromosomalelements called plasmids. Most simply, plasmids can beconsidered as primitive bacteriophages that carry a function that allowsthe unit to be replicated autonomously (the replication system), but thatlack the genetic information required for a complex life cycle or existencein an extracellular state (Cohen, 1976). Circular plasmid DNA molecules(Fig. 1) are physically separate from the bacterial chromosome, andthey can encode a variety of genetic traits that are not essential forgrowth of the host cell but that commonly provide a biological advantageto cells carrying the plasmids; antibiotic resistance is one of these properties.Examples of other traits carried by plasmids are shown in Table I.Plasmids commonly are present in multiple copies within each cell,and plasmid DNA preparations isolated from bacterial cultures contain aheterogeneous population of DNA molecules. To employ classicalgenetic methods for the study of plasmid mutants, and to investigate theorganization of genetic information on plasmid DNA, it was thereforenecessary to establish a method for the cloning of individual plasmidDNA molecules. Procedures for transforming bacteria for chromosomallyencoded traits had been developed for Pneumococcus,Haemophilus, Bacillus, and certain other organisms (Avery et al.,1944; Hotchkiss and Gabor, 1970), but transformation had not beenshown for Escherichia coli or the other enteric bacteria with which wewere working. It was known that treatment of E. coli cells with calciumTRANSPLANTATION OF GENESFIG. 1. Electron photomicrograph showing twisted "supercoiled" and "open" circularm6lecules of the antibiotic resistance plasmid, R1. From Cohen and Miller (1969).TABLE 1SOME PROPERTIESE NCODED BY NATURALLYOCURRINGPLASMIDS"Antibiotic resistanceFertility (ability to transfer genetic material by conjugation)Production of bacteriocinsAntibiotic productionHeavy-metal resistance (Cd2+, HgZ+ )Ultraviolet resistanceEnterotoxinVirulence factors, hemolysin, K 88 antigenMetabolism of camphor, octane, and other polycyclic hydrocarbonsTumorigenicity in plantsRestriction/modification" Modified from Cohen (1976).176 STANLEYN. COHENchloride enabled them to take up DNA of the bacteriophage A, and thatviable viral particles were produced in such CaC1,-treated bacteria;however, attempts to generate clones that had acquired new geneticproperties from the transformed DNA had not been successful (Mandeland Higa, 1970).In 1972, my colleagues and I found, using a modification of thepreviously described CaClz procedure, that E. coli could take up circularplasmid DNA molecules (Fig. 2), and that a line of transformed cellsthat phenotypically express genetic information carried by the incomingplasmid DNA could be produced (Cohen et al., 1972). While this wasan inefficient process (approximately one in 106 cells were transformed),transformants could readily be identified and selected by utilizingthe antibiotic resistance genes carried by the plasmids we werestudying. Plasmid-transformed cells reproduced themselves normally,and acquired a DNA species having the same genetic and molecularproperties as the parent plasmid. Since each cell in the resulting clonecontains a replica of the single plasmid DNA molecule that was taken upTETRACYCLINE-RESISTANT TOTAL CELL DNABACTERIAL CELL + ETHlDlUM BROMIDECHROMOSOMALDNA EXTRACTIONCHROMOSOME 4)- CESIUM 1CHL ORICD-EEN TRIFUGATION ~0 FR~A CTIONpA DTNIOAN L ~Tc RESISTANCEPLASMIDPERMEABLE 'COMPETENT" CHROMOSOMALDNATETRACYCLINE SENSITIVEBACTERIAL CELLTc RESISTANCECHROMOSOMETRANSFORMED Tc RESISTANTBACTERIAL CELLFIG. 2. Schematic presentation of plasmid DNA transformation procedure. Purifiedplasmid DNA separated from chromosomal DNA by cesium chloride-ethidium bromidegradient centrifugation is i n t r o d u c e d into bacteria made permeable to DNA by treatmentwith calcium chloride. Antibiotic r e s i s t a n c e genes carried by the plasmid are used in theselection of transformed bacterial cells.~ ~ DTRANSPLANTATION OF GENES 177by the originally transformed bacterium, the procedure made it possibleto clone (and thus separate and purify biologically) genetically distinctplasmids present in a heterogeneous population. We could now apply tothe study of plasmids a variety of genetic and biochemical methods thatpreviously were restricted to bacteriophages, which could be clonedbecause of their plaque-forming properties.To determine the genetic and molecular properties of specific regionsof the DNA of large antibiotic resistance plasmids (R-plasmids), webegan to take these plasmids apart by shearing the molecules mechanicallyand then introducing the resulting DNA fragments into CaC1,-treated E. coli cells by transformation (Cohen and Chang, 1973). However,work being carried out with restriction endonucleases in otherlaboratories (Smith and Wilcox, 1970; Kelly and Smith, 1970; Dannaand Nathans, 1971) suggested that these enzymes would be highlyuseful in our analysis. It had been discovered that restriction endonucleases,which are produced by many different species of bacterial cells,can recognize specific nucleotide sequences within DNA and can cleaveDNA molecules at these recognition sites (Smith and Wilcox, 1970;Kelly and Smith, 1970). The cell's own DNA is protected from cleavageby modification enzymes (methylases) that add methyl groups tocertain nucleotides within the recognition sequence, rendering the siteresistant to cleavage by the companion endonuclease (Arber, 1965;Meselson and Yuan, 1968; Nathans and Smith, 1975). Thus, restrictionendonucleases could be used to generate reproducibly a characteristicset of cleavage fragments for each plasmid; for most of our experiments,this would be preferable to generating a random series of plasmid DNAfragments by mechanical shearing. Moreover, the fragments producedby restriction enzyme cleavage could be analyzed and characterized byelectrophoresis on gels; such methods had already been used effectivelyby Nathans and his collaborators for analysis of the SV40 animal virusgenome (Danna and Nathans, 197 1;Nathans and Danna, 1972).B. The Joining of Separate DNA Fragments in VitroThe conceptual and experimental basis for linking DNA segments bymeans of projecting single-strand ends having complementary nucleotidescan be found in the work of Khorana and his collaborators, whoin the late 1960s showed that short segments of synthetic DNA could be178 STANLEY N. COHENjoined by the addition of overlapping complementary single-strandedsegments (Khorana, 1968; Agarwal et al., 1970). The construction ofsuch complementary DNA sequences by the addition of single nucleotideswas laborious, however. Jensen et al. (1971) first reported theuse of the enzyme terminal transferase to add homopolymeric stretchesof deoxyadenosine (dA) or deoxythymidine (dT) to the ends of DNAfragments in an attempt to link the fragments covalently in vitro by (a)hydrogen bonding of the complentary nucleotides; ( 6 )subsequent closureof the resulting single-strand breaks by DNA ligation. In theseconceptually sound, but only partially successful experiments, a seriesof DNA molecules were joined together end-to-end by dA-dT "tails" toform catenated structures; however, Jensen et al. did not achieve thefinal step (i.e., ligation) necessary to accomplish covalent DNA linkage.It is now known that in vitro ligation is not required for thecovalent joining of separate DNA segments that contain homopolymericadditions; ligation of such segments occurs in vivo when the hydrogenbondedsegments are introduced into bacterial cells by transformation(Wensink et al., 1974).The problem of in vitro ligation of DNA fragments that havehomopolymeric extensions at their ends was solved by the discovery byLobban and Kaiser (1973) that such covalent joining could be achievedby the use of exonuclease 111, and this finding was employed by Jacksonet al. (1972) in linking the tumor virus SV40 to DNA molecules ofbacteriophage Adv. It has been of some historical interest that concernabout possible biohazards related to the SV40 component of the hybridAdv-SV40 molecule that Jackson et al. had constructed led Berg and hiscolleagues to decide not to try to clone the molecule in E. coli (Wade,1974). Ironically, however, with regard to the biosafety controversythat ensued (Berg et al., 1975), we can reasonably assume that nobacterial clones carrying the composite molecule would have resulted ifthe experiment had been tried: the Adv cleavage site at which the twoDNA segments were joined is located within a gene essential for replicationof Adv, and interruption of the continuity of this gene by an insertedDNA fragment prevents the bacteriophage DNA molecule fromfunctioning as a replicon (Helling et al., 1974; Streek and Hobom,1975; Mukai et al., 1976).The subsequent discovery that restriction endonucleases could generatein one step DNA termini having projecting single-strand ends, andTRANSPLANTATION OF GENES 179that these could be linked to a complementary nucleotide sequence onanother endonuclease-generated DNA fragment, made the joining ofDNA segments much simpler. The nucleotide sequences that constitutethe cleavage sites for several endonucleases were identified in the early1970s; in every instance, cleavage occurred at or near an axis of bidirectionalrotational symmetry: that is, the endonuclease recognition siteconsists of a sequence that reads the same on both DNA strands in the 5'to 3' direction. Often, restriction endonucleases cleave both DNAstrands at precisely the same location, yielding blunt-ended DNA fragments(for review, see Nathans and Smith, 1975). Certain of theseendonucleases, however (for example, the EcoRI enzyme), introducebreaks that are several nucleotides apart in the two DNA strands (Fig.3). Because of the bidirectional rotational symmetry of the nucleotidesequence in the region of cleavage, cleavage of the two DNA strands atseparated points within this region yields fragments that have protrudingcomplementary nucleotide sequences at their ends. Such termini, whichresemble mortise and tenon type joints, can be linked together by hydrogenbonding. Since all DNA termini generated by the enzyme areCIRCULARDNAMOLECULE1-Eco RI ENDONUCLEASE CLEAVAGEFIG.3. The six-nucleotide-long recognition sequence cleaved by the EcoRI endonucleaseis shown. Because of the bidirectional rotational symmetry of the nucleotide sequencein the region of the cleavage, the two DNA strands are cut at separate points,yielding fragments that have protruding complementary single-strand ends.180 STANLEY N.COHENidentical, fragments derived from different DNA molecules can bespliced together.The finding that the DNA fragments generated by the EcoRT restrictionendonuclease have projecting single strands at their termini wasreported simultaneously in 1972 by Sgaramella (1972) and by Mertz andDavis (1972). Sgaramella found that molecules of the bacterial virusP22 cleaved with the EcoRI enzyme can form catenated DNA segmentsequal in length to two or more viral DNA molecules. Mertz and Davisobserved that closed-loop SV40 DNA molecules cleaved by EcoRIcould re-form themselves into circular molecules by hydrogen bondingand could be sealed covalently with DNA ligase; furthermore, the reconstitutedmolecules were infectious in animal cells growing in tissueculture. While this property of the EcoRI enzyme and certain other restrictionendonucleases was of great importance in the development ofrecombinant DNA methods, it is now appreciated that cohesive DNA terminiare not essential for the linkage of DNA termini. Sgaramella et al.(1970) had reported that even blunt-ended DNA fragments can bejoined together by use of the bacteriophage T4 ligase; such blunt-endedjoining has found widespread use in the linking together of DNAfragments generated by restriction endonucleases that do not yieldprojecting single-strand ends (Sgaramella et al., 1977), and for thejoining of DNA fragments that have been made blunt-ended by the S1nuclease or DNA polymerase I (Bolivar et al., 1977; Chang andCohen, 1978).The discovery of DNA ligases (Gellert, 1967; Weiss and Richardson,1967; Gefter et al., 1967; Olivera and Lehman, 1967; Cozzarelli et al.,1967) also has had a major role in the development of recombinantDNA methods. These enzymes, which can form phosphodiester bondsbetween adjacent DNA nucleotides, are required for the in vitro joiningof DNA molecules. However, as noted above it is now known that invitro ligation is not necessary to join DNA fragments that are being heldtogether by extended homopolymeric terminal additions (Wensink etal., 1974). Fragments that have protruding single-strand ends generatedby restriction endonucleases can also be linked together in vivo by theintracellular action of DNA ligase (Mertz and Davis, 1972; Cohen etal., 1973), and such linkage can fully and accurately reconstitute thegenetic continuity of the DNA sequence (Chang and Cohen, 1977).-..aTRANSPLANTATION OF GENES 181C. Construction of Biologically Functional Bacterial Plasmids in VitroTo determine whether large and complex plasmid DNA moleculescould be reduced in size or restructured entirely by cleaving them intomultiple fragments with a restriction endonuclease and joining togetherthe resulting fragments in a different arrangement, A. C. Y. Chang,H. W. Boyer, R. B. Helling, and I studied the large antibiotic resistanceplasmid R6-5 (Cohen et al., 1973). We established that this plasmid(Silver and Cohen, 1972), which consists of almost 100,000 nucleotidebase pairs and contains several genes encoding several different antibioticresistances, was cleaved into 11 separate DNA fragments by theEcoRI endonuclease; hopefully the location of the cleavage siteswould leave the replication machinery of the plasmid and one ormore of its antibiotic resistance genes intact. R6-5 DNA was treatedwith the EcoRI enzyme and was introduced by transformation intoCaC1,-treated E. coli cells with or without prior ligation of the DNA.Selection was carried out for transformants that expressed one or moreof the antibiotic resistance determinants located on the parent plasmid.One such clone, which expressed kanamycin (Km) resistance butnone of the other antibiotic resistances of R6-5, was identified and itsplasmid DNA was isolated and characterized by EcoRI endonucleasedigestion and agarose gel electrophoresis (Fig. 4). The digestion patternshowed that a new plasmid replicon containing only 3 of the 11 EcoRIfragments of R6-5 had been formed. By selecting for propagation of theKm resistance gene of R6-5, we had been able to clone a specific DNAsegment carrying this gene. The Km resistance fragment, which welater showed does not have the capacity for autonomous replication, hadbecome linked to an EcoRI-generated DNA fragment carrying the rep-FIG.4. Agarose gel electrophores of EcoRI digest of the pSC102 plasmid (A) containingthree of the EcoRI-generated fragments comprising the R6-5 plasmid (B).ThepSClOl plasmid is cleaved by the EcoRI endonuclease only once to yield a single linearDNA fragment (C). From Cohen er al. (1973).182 STANLEY N. COHENlication region of R6-5, and this enabled its propagation in transformedbacteria (Cohen et al., 1973). These findings demonstrated that a plasmidDNA segment carrying replication functions could serve as a cloningvehicle or "vector" for the cloning of other restrictionendonuclease-generated DNA fragments. Ideally, a plasmid vector suitablefor the cloning of nonreplicating EcoRI-generated DNA fragmentswould contain replication machinery plus a selectable antibiotic resis- -tance gene on the same EcoRI fragment. We searched for such a vectoramong the antibiotic resistance plasmids we had been studying.In our collection at Stanford was a small plasmid, 9000 base pairs inlength, that carried a gene conferring resistance to the antibiotic tetracycline(Tc). When we subjected the DNA of this plasmid (pSC 10 1)(Cohen and Chang, 1973, 1977) to cleavage by EcoRI endonucleaseand analyzed the products by gel electrophoresis, we found that theenzyme had cut the DNA molecule at only a single location. Thisindicated that the pSClOl plasmid could be used as a directly selectablecloning vector if a fragment of foreign DNA could be inserted at itsEcoRI cleavage site without interfering with either the replicationfunctions or expression of the Tc resistance gene carried by the plasmid.We mixed the DNA of the pSClOl plasmid with the previouslyconstructed R6-5-derived plasmid carrying a Km resistance gene on anEcoRI-generated fragment, cleaved the mixture with EcoRI endonuclease,and treated the resulting DNA with ligase. The DNA was introducedinto E. coli by transformation, and bacteria that expressed boththe R6-5-derived Km resistance determinant and the Tc resistance geneof pSClOl were selected. A plasmid from one of the resulting cloneswas found to contain the entire pSClOl vector plus one of the threefragments of the Km-resistance plasmid (Fig. 5). Thus, pSCl 01 couldat least be used to propagate a nonreplicating segment of another EcoRI ,DNA plasmid. In similar experiments, we showed that the pSClOlplasmid could be joined in vitro to a second EcoRI-cleaved repliconcarrying a gene for streptomycin resistance. The procedure is sum- --marized schematically in Fig. 6.Chang and I proceeded to determine whether the procedure we hadused to clone fragments of E. coli plasmids could be used to propagateand genetically express DNA from an unrelated bacterial species (Changand Cohen, 1974). It was possible that the way genetic information wasarranged on totally foreign DNA molecules or another yet unknownTRANSPLANTATION OF GENES 183FIG.5. Agarose gel electrophoresis of EcoRI digest of newly constructed plasmidDNA species. A new plasmid (A) consisting of the pSC101 vector (D) plus the kanamycinresistance (middle) fragment of pSC102. (C) has been constructed by EcoRI cleavage ofthe parental DNA molecules plus ligation and &ansformation. (B) shows a mixtureof the EcoRI-cleaved plasmid DNA preparations. From Cohen et al. (1973).factor might produce an aberrant situation that would prevent the survivalof such hybrid molecules in a new host. [It is now known thatthe DNA sequence arrangement on some DNA fragments impedes theircloning or stability, or both, as part of recombinant DNA molecules(Heyneker et al., 1976; Timmis et al., 1978b)l. Even if DNA from avery different bacterial species, such as Staphylococcus aureus, couldbe replicated in E. coli by joining it to the pSClOl vector, the foreigngenes might not be expressed phenotypically in a heterospecific environment.[There is now evidence that some genes derived from foreignbacterial species can be expressed phenotypically in E. coli, but otherscannot (Chakrabarty et al., 1978); we made a fortunate choice in selectinga gene that was expressed.]EcoRI-cleaved pSClOl plasmid DNA and DNA from the S. aureusplasmid p1258, which carries a gene that encodes the enzyme/3-lactamase and specifies resistance to penicillin and ampicillin (Ap),-- were mixed, treated with DNA ligase, and introduced into E. coli bytransformation. Transformant cells that expressed the penicillin resistanceof the S. aureus plasmid as well as the Tc resistance of E. coliwere isolated; these were found to contain a new DNA species consistingof the entire pSClOl plasmid plus an EcoRI-generated S. aureusDNA fragment that contained the Ap resistance gene derived from thepI258 plasmid (Fig. 7).0STANLEY N. COHENnSClO1 PLASMIDREPLICATOR LE~;A;GcEo RI FOREIGN DNAEco RI ENDONUCLEASEANNEALING-TRANSFORMATIONTRANSFORMEDPLASMIDFIG. 6. Schematic representation of the procedure used in the initial DNA cloningexperiments. Fragments of EcoRI endonuclease-cleaved DNA were joined to the similarlycleaved pSClOl plasmid vector by hydrogen bonding of protruding single strandscontaining complementary base sequences. After covalent joining of the fragments byDNA ligase, they were introduced by transformation into CaC1,-treated bacteria. Cellsresistant to tetracycline were selected, and each yielded a bacterial clone containing aplasmid identical to the pfasmid DNA molecule taken up by a single transformed cell.The replication and expression in E. coli of genes derived from anorganism not known to exchange DNA with E. coli suggested that =interspecies genetic combinations might be generally obtainable. Wereasoned that it might be practical to use these methods to introduce intoE. coli genes specifying metabolic and synthetic functions indigenous toother biological classes. Potentially, plasmid replicons such as pSClOlmight also allow DNA derived from eukaryotic organisms to be introducedinto E. coli, thus enabling the application of bacterial genetic andTRANSPLANTATION OF GENES 185biochemica1,techniques to the study of eukaryotic genes. Moreover, byfragmenting the eukaryotic chromosome and cloning segments of it onindividual plasmids, it potentially would be feasible to isolate specificeukaryotic genes and to study the organization of genetic information ofhigher organisms in ways that were not previously possible.DNA EX1EcoRl CLEAVAGE EcoRl SITE - AP - II Rep( EcoRl CLEAVED I I TRANSFORMATION TO E.coli K12SELECTION FOR ~ p 'FIG.7. Chimeric plasmids containing DNA segments derived from Staphylococcusaureus and Escherichia coli were constructed by joining an EcoRI-generated fragmentfrom the S. aureus plasmid pI258 to the pSClOl vector and introducing the compositemolecule into E. coli. The Ap-resistance gene canied by the S. aureus plasmid DNA wasexpressed phenotypically in the unrelated bacterial host.186 STANLEY N.COHEND. Cloning of Eukaryotic DNA in E. coliTo determine whether eukaryotic DNA could in fact be replicated inbacteria, my colleagues and I undertook the cloning of DNA that encodesthe ribosomal RNA of the frog Xenopus laevis (Morrow et al.,1974). Although this DNA does not express traits (such as antibioticresistance) that enable selection of bacteria canying chimeric plasmids, -X. laevis ribosomal DNA (rDNA) had been well characterized, and itsphysical properties would permit the identification of X. laevis DNAfragments of bacterial plasmids. The Tc resistance conferred by thepSClOl plasmid allowed us to select for transformed clones, and wecould then examine the plasmid DNA isolated from such clones todetermine whether any of the plasmids contained DNA fragments havingmolecular properties of Xenopus ribosomal DNA. The foreign DNAfragments being propagated in bacteria could also be tested for nucleotidesequence homology with DNA isolated directly from X. laevisoocytes, using electron microscope heteroduplex techniques (Davis andDavidson, 1968; Westmoreland et al., 1969).Ribosomal DNA from X. laevis and the pSClOl plasmid weremixed, cleaved with EcoRI endonuclease, and ligated using the procedureswe had employed earlier. Fifty-five Tc-resistant transformantswere isolated, and DNA obtained from such transformants was analyzedby gel electrophoresis, cesium chloride gradient centrifugation, and/orelectron microscopy to determine the presence of an EcoRI-generatedDNA fragment similar in size and/or buoyant density to similarly generatedfragments of bona fide X. laevis rDNA. 'The results of these experimentsare summarized in Table 11. Fifteen of the Tc-resistance clonescontained one or more EcoRI-generated fragments having the same sizeas fragments produced by cleavage of X. laevis rDNA. Moreover, theplasmid chimeras isolated from E. coli were shown to contain DNAwith a buoyant density characteristic of the high G+C base compositionof X. laevis rDNA. These experiments also produced an unexpected =finding that provided an example of the type of new information thatDNA cloning procedures could yield about the organization and structureof eukaryotic chromosomes. Variation in size of the EcoRIgeneratedX. laevis rDNA fragments present in plasmid chimeras wasobserved; together with the EcoRI cleavage pattern found in theamplified X. laevis rDNA isolated from frog oocytes, this findingTRANSPLANTATION OF GENES 187TABLE 11Xenopus laevis-Escherichia coli RECOMBINANTPLASMIDS"'~Molecular weightof EcoRl plasmidfragments Molecular weight Buoyant densityestimated by from contour of intact plasmidgel electrophoresis length in CsClPlasmid DNA (x lo-=) (x lo-=) (p/cm3)pSClOl 5.8 6.0 1.710" Modified from Morrow et al. (1974).EcoRI-cleaved chimeric plasrnids containing X. laevis rDNA were characterized bybuoyant density centrifugation in cesium chloride, electron microscopy, and electrophoresisin agarose gels.suggested that the amplified repeat unit was heterogeneous in the oocytes(Morrow et al., 1974).Electron microscope analysis (Fig. 8) of a heteroduplex formed betweenX. laevis rDNA and one of the plasmid chimeras (CD42)demonstrated that this plasmid contains DNA nucleotide sequenceshomologous with those present in rDNA isolated directly from X.laevis. In some instances, segments of two separate chimeric plasmidDNA molecules were seen to form duplex regions with the single strandof X. laevis rDNA, consistent with the observation (Dawid et al., 1970;.- Wensink and Brown, 1971) that the rDNA sequences of amplified X.laevis are tandomly repeated.The plasmid chimeras containing both E. coli and X . laevis rDNAwere found to replicate stably in bacterial hosts as part of the pSCl0lplasmid replicon and could be recovered from transformed E. coli byprocedures commonly employed for the isolation of bacterial plasmids.Tritium-labeled RNA isolated from bacteria harboring these plasmids188 STANLEY N. COHENFIG. 8. Electron photomicrograph of a heteroduplex of Xenopus laevis ribosomal DNAand two separate molecules of a tetracycline resistance plasmid chimera (CD42) isolatedfrom E. coli and containing a cloned DNA fragment derived from X. laevis. A, SinglestrandrDNA X. laevis; B, double-strand regions of homology between the plasmid andX. luevis rDNA; C, single-strand segments corresponding in length to the DNA segmentof the plasmid derived from the pSClOl plasmid vector. From Morrow er al. (1974). ..hybridized in vitro to amplified X. laevis rDNA isolated directly fromthe eukaryotic organism, indicating that RNA synthesis could occur on --the eukaryotic DNA transplanted into the prokaryotic host.Since these early DNA cloning experiments, major advances made ina number of laboratories have increased the ease and flexibility of geneTRANSPLANTATION OF GENES 189manipulation, so that segments of DNA molecules can now be takenapart and put together in a variety of different ways. Dozens of sitespecificendonucleases that recognize different nucleotide sequencesand thus cleave DNA at different sites have been identified and characterized(Roberts, 1976). Synthetic and natural "adaptor" fragmentshave been used to convert one kind of endonuclease cleavage site to- another (Marians et al., 1976; Heyneker et al., 1976; Cohen et al.,1977; Roberts, 1977; Scheller et al., 1977). Additional naturally occurringplasmids suitable as vectors were identified (Hershfield et al.,1974), and recombinant DNA methods have been used to modify theseplasmids to yield vectors suitable for specific purposes (Armstrong etal., 1977, Timmis et al., 1978c; Bolivar et al., 1977; Chang andCohen, 1978). Vectors that utilize the replication and packaging systemsof bacteriophage A (Rambach and Tiollais, 1974; Murray andMurray, 1974; Thomas et al., 1974; Blattner et al., 1977; Leder et al.,1977; Hohn and Murray, 1977) or other bacteriophages (Messing et al.,1977; Hermann et al., 1978). Specific messenger RNA (mRNA)species produced by certain organs or tissues has been used as templatefor the enzymic synthesis of double-stranded complementary DNA(cDNA) sequences corresponding to the mRNA (Ruogeon et al., 1975;Rabbits, 1976; Eftratiadis et al., 1976). Double-stranded DNA segmentsthat have a nucleotide sequence corresponding to a known aminoacid sequence have been synthesized chemically and have been purifiedand amplified by cloning them as part of a bacterial plasmid (Itakura etal., 1977; Goeddel et al., 1979). Novel methods of detecting plasmidsthat include specifically desired gene sequences have been developedusing subculture cloning procedures (Kedes et al., 1975) or in situhybridization procedures (Grunstein and Hogness , 1975). Cotransformationprocedures that enable introduction of nonselectable segments ofDNA into bacteria (Kretschmer et al., 1975) or mammalian cells(Wigler et al., 1977) have been devised.Although the site-specific endonucleases used for gene manipulationin vitro are commonly called "restriction enzymes," some of the bacterialspecies that encode such endonucleases show no detectable restrictionof foreign DNA in vivo, and it has been speculated that the primaryfunction of such enzymes may be DNA recombination (Kornberg,1974; Nathans and Smith, 1975; Roberts, 1976). It seems highly likelythat DNA cleavage by at least some restriction endonucleases also oc190STANLEY N. COHENcurs in vivo: the transforming ability of infecting phage DNA is restrictedby several orders of magnitude in cells that produce the EcoRIenzyme (Takano et al., 1968a,b), implying that most of the enteringDNA molecules are cleaved in vivo before they can be methylated bythe modification enzyme associated with the EcoRI restrictionmodificationsystem. There is evidence that the combined actions of theEcoRI endonuclease and DNA ligase can promote site-specific recombinationin vivo, with results similar to the effects of these enzymesin vitro (Chang and Cohen, 1977). Moreover, "transposons, " whichcan operate in vivo to join DNA segments having no ancestral relationship,can accomplish a result that is analogous to in vitro site-specificrecombination (Cohen, 1976).IV. USEOF DNA CLONINAGS A TOOLFOR THE STUDYO FPROKARYOTAINCD EUKARYOTBICIO LOGYA . Studies of Plasmid BiologyThe wish to study bacterial plasmids themselves was the motive thatinitially prompted our development of DNA cloning methods, and duringthe past 6 years my laboratory has used these methods extensively insuch studies. DNA cloning has made possible elucidation of the structureand control of plasmid genes and has yielded much informationabout the replication of plasmid DNA. Using nonreplicating DNAfragments that contain antibiotic resistance genes as biological "probes, "it has been possible to isolate and study DNA fragments carrying thereplication functions of large and structurally complex plasmids (forexample, Timmis et al., 1975; Lovett and Helinski, 1976; Taylor and . Cohen, 1979), as well as those of small plasmids (Chang and Cohen,1 978) (Fig. 9).Using hybrid replicons formed by the fusion of two functionally *-different types of replication systems, we have investigated the relationshipof plasmid replication and incompatibility (Timmis et al., 1974;Cabello et al., 1976; Meacock and Cohen, 1979) and have studiedreplication control in plasmids. A DNA sequence that accomplishesactive partitioning of plasmids in dividing cell populations and that isfunctionally equivalent to the centromere of eukaryotic cells has beenNon- replicating€LORI fragmentEcoRIDigest ion-FRACTION NUMBERE 2 R I d~gestof complex repliconLigation and -TransformationFIG. 9. Scheme for isolation of replication regions of complex plasmids. In the experiment shown, a plasmidcarrying a nonreplicating Ap-resistance segment was cleaved by the EcoRI restriction endonuclease, and the Apresistance"probe" fragment was separated from its vector. The probe was then added to a mixture of DNA fragmentsproduced by EcoRI cleavage of a large plasmid, and ligation and transformation were canied out. Since theprobe fragment is incapable of replication, its propagation in transformants requires linkage to a DNA segmentcarrying replication functions. From Timmis el al. (1978~).192 STANLEY N. COHENdiscovered and characterized using DNA cloning methods (P. Meacockand S. N. Cohen, unpublished data). The genes carried by large antibioticresistance plasmids have been assigned to specific loci on plasmidDNA by the cloning of endonuclease-generated DNA fragments, andmaps of complex plasmid genomes have been constructed (for example,see Timmis et al., 1978a). Natural evolutionary variations in plasmidstructure have been identified and have led to the concept that plasmid .DNA is in a constant state of flux undergoing both macro- and microevolution(Chang et al., 1975; Brutlag et al., 1976; Cohen et al., 1978;Timmis et al., 1978b). Genes within transposable genetic elementshave been studied, and their functional interactions have been elucidated.B . Study of Organization of the Eukaryotic Genetic Sequence EncodingPro-opiocortinWe and others have also used DNA cloning methods for the study ofgene organization, evolution, and expression in eukaryotes. Of particularrecent interest to my laboratory has been the genetic sequence thatencodes the pituitary hormones ACTH and /3-lipotropin @-LPH). Thesepeptide hormones are known each to include smaller peptides havingdistinct biological activities: a-melanotropin (a-MSH) andcorticotropin-like intermediate lobe peptide (CLIP) are derived fromACTH; /3-melanotropin @-MSH), endorphins, and methionine enkephalinare included within /3-LPH (Scott et al., 1973; Li and Chung,1976; Ling et al., 1976; Li et al., 1977) (Fig. 10). The intracellularlevel of the mRNA encoding the common precursor protein (proopiocortin)is known to be depressed by glucocbrticoids, which seem toact at the transcriptional level by means of a glucocorticoid receptor(Nakanishi et al., 1977; Nakamura et al., 1978). The various componentpeptides are liberated from pro-opiocortin and secreted frompituitary cells by processing mechanisms.Although the general positions of ACTH and /3-LPH on the proopiocortinpeptide have been known for several years, earlier studieshad provided no information about the precise relationships of thesepeptides and the nature of the processing that the precursor moleculeundergoes to yield its two major components. Moreover, ACTH and/3-LPH account for only one-third to one-half of the molecular weight ofTRANSPLANTATION OF GENES-+4 Precursor protein39 Amino acids 93 Amino acids-114 ,I"cryptic" region - 1 - i Y-LPH MET-ENKEPHALINFIG.10. Map of pro-opiocortin (corticotropin-P-lipotropin)precursor protein showingpeptide components previously identified by amino acid analysis and "cryptic" region.Corticotropin (ACTH) and P-lipotropin (P-LPH) were positioned on pro-opiocortin byanalysis of cloned cDNA derived from mRNA encoding the precursor protein. The lengthshown for P-LPH (93 amino acids) has been assigned from the nucleotide sequence of acloned cDNA insert and differs from the commonly accepted 91 amino acid sequence forP-LPH determined by amino acid analysis (Li et al.. 1977).the precursor protein; thus there has been considerable interest in, andspeculation about, the primary structure and possible biologicalfunctions of the peptides encoded by the remaining "cryptic" portion.Our recent studies of the genetic sequence encoding pro-opiocortin providean example of the application of DNA cloning methods for theinvestigation of gene organization in eukaryotgs.The cloning of complementary DNA (cDNA) (Nakanishi et al.,1977) corresponding to the sequence encoding mRNA the ACTH-PLPHprecursor protein was carried out utilizing mRNA purified fromthe neurointermediate lobe of bovine pituitaries (Kita et al., 1979).Avian myeloblastosis virus (AMV) reverse transcriptase was used forthe sequential synthesis of the two strands of cDNA, homopolymeric dC"tails" were added, and complementary poly(dG) extensions were.* added to PstI endonuclease cleaved-DNA of the Tc resistance plasmidvector pBR322 (Bolivar, 1977). These steps are summarized in Fig. 1 1.Following transformation of E. coli cells with the dG-dC tailed proopiocortincDNA, Tc-resistant transfomants were isolated, and bacterialclones that contained cDNA inserts were identified by a colonyhybridization procedure (Grunstein and Hogness, 1975) using 32Plabeledpituitary mRNA as a probe.STANLEY N.COHENmRNA TI Plasmid pBR 322 AAAAIIJIReverseTranscriptaseAAAAAC T T T T T 4Alkali Digestion7 Pst I Endonuclease IReverseTranscriptase aCGTCG G ' CTGCA ... - b~erminal- 1 TransferaseG CTGCPGGGG NuClease GGGGACGTC GTerminalTransferaseccccCCCC /GGGGACGTCC TGCAGGGGTransformationFIG. I I. Outline for protocol used for cloning of pro-opiocortin mRNA. For details,see text and Nakanishi et a/. (1979). The Tc resistance gene on plasmid pBR322 was usedfor selection of transfonnants. As shown in the figure, the recognition sequence for PstIendonuclease is regenerated at the plasmid/cDNA junction by the "tailing" procedure - used.The plasmid present in one of these clones (pSNAC20) was selected *-for further study. By determining the entire 1091 base pair nucleotidesequence (Maxam and Gilbert, 1978) of the cDNA insert of thepSNAC20 plasmid, we were able to infer certain important features ofthe protein encoded by the pro-opiocortin mRNA. Since the amino acidcomposition of ACTH and ,B-LPH are known (Scott et al., 1973; Li andChung, 1976; Ling et al., 1976, Li et al., 1977), the translationalTRANSPLANTATION OF GENES 195reading frame of the cDNA sequence could be determined, and anamino acid sequence could thus be assigned for the previously crypticsegment of the pro-opiocortin protein. A probable translational initiationcodon (AUG) for the precursor protein was identified from thetranslational reading frame and the previously known approximatelength of pro-opiocortin. The first 20 amino acid residues following theputative initiative methionine were found to include a large proportionof hydrophobic amino acids (13 nonpolar residues, including 7leucines), consistent with a putative role for the amino-terminal segmentof pro-opiocortin as a "signal" peptide (Blobel and Dobberstein,1975a,b) involved in secretion of the protein. This assignment has beenverified recently by analysis of peptide fragments derived from thepreviously cryptic segment of the protein (Nakamura er al., 1979;Keutmann et al., 1979; E. Herbert, personal communication).Computer analysis of amino acids assigned from the DNA sequenceof the cryptic portion of the precursor protein showed that the proopiocortinprotein contains a sequence of amino acids strikingly similarto the amino acid sequences of the previously identified hormonesa-MSH and P-MSH. As in the case of a - and P-MSH, this peptidesegment (which was named y-MSH, Nakanishi et al., 1979) is flankedby pairs of the basic amino acids lysine and/or arginine, suggesting thatit could be liberated from pro-opiocortin by proteolytic processing. Asecond peptide segment located within the putative signal peptide segmentof pro-opiocortin was found to have less extensive structural similarityto the MSHs; the presence of several largely homologous unitswithin the same precursor molecule (Fig. 12) suggests that the gene forpro-opiocortin may have been formed by a series of structural duplications.The previously "cryptic" part of the pro-opiocortin molecule was.. also found to contain a number of amino acids in positions equivalent tothose found in the hormone calcitonin, which is believed to have biologicalfunctions quite unrelated to those of the other components of-' molecule (Chang et al., 1979).Recently, we have isolated plasmids that include genomic DNA sequencesencoding for human pro-opiocortin. Comparison of the DNAsequence of such clones with the cDNA sequence for the bovine hormoneshould provide information about the extent of interspecies variationwithin the cryptic part of the molecule and may yield data relatingthe ACTH and P-LPH coding sequences on the human chromosome to----196 STANLEY N.COHEN-120 -100 -80 -60 -40 -2; -1 1 20 40 60 80 100 120I I I I I \ f l I I I I I- V//AFutative 7-MSH ACTH ( I - 39) D-LPH (42-134)SignalPeptlde d-MSH CLIP 7-LPH 0-Endorphin(1-13) (18-39) (42-101) (104.134)I0-MSH Met-Enkephalin(84-101) (104-108)FIG. 12. Schematic representation of the structure of bovine pro-opiocortin. The numberingof the amino acid residues is as described in Nakanishi et a[. (1979). Filled barsrepresent the region for which the amino acid sequence was known independently, and theopen and hatched bars represent the regions for which the amino acid sequence waspredicted from the nucleotide sequence of the pro-opiocortin mRNA. The Lys-Arg residuesat sites of possible processing of the precursor protein into its peptide components,the positions of amino acids relevant to the MSH-like subunits of the protein, and certainother structural features are indicated. From Nakanishi er al. (1979).the genes encoding calcitonin and other hormones. It should also provideinsight into the relationship of intervening sequences to theprotein-encoding sequences comprising the various structural andfunctional domains of the precursor protein.C. Expression of Mammalian DNA Sequences in Bacterial Cells -Since the initial propagation of eukaryotic DNA in bacteria (Morrowet al., 1974), several systems have been used to study expression in E.coli of DNA derived from higher organisms. Our early studies with '-cloned X. laevis ribosomal DNA genes indicated that the nucleotidesequences of the eukaryotic DNA could be faithfully transcribed in E.coli (Morrow et al., 1974). However, these experiments did not showwhether such RNA synthesis resulted from read-through transcriptionfrom the bacterial component of the chimeric plasmids or from initiationof RNA synthesis on the eukaryotic DNA fragment. Subsequent invesTRANSPLANTATIONOF GENES 197tigations with plasmids containing the intact mouse mitochondria1 DNAgenome (Chang et al., 1975) indicated that the transcriptional and translationalcontrol signals located on at least this eukaryotic cell-derivedDNA did not function in bacteria to yield bona fide eukaryotic proteins.Biological activity of genes from the lower eukaryotes Saccharomycescerevisiae and Neurospora crassa was demonstrated sub- - sequently using phenotypic selection for functions that complement mutationallyinactivated homologous bacterial genes (Struhl et al., 1976;Ratzkin and Carbon, 1977; Vapnek et al., 1977). Later, immunologicalactivity with antibody against the human hormones somatostatin andinsulin was shown for peptide fragments cleaved in vitro from hybrid"fusion" proteins encoded in part by bacterial DNA and in part bychemically synthesized somatostatin or insulin DNA sequences (Itakuraet al., 1977; Goeddel et al., 1978). In another instance, a hybrid proteincontaining the amino acids of proinsulin was shown to be made bybacteria that carry a double-stranded cDNA transcript of preproinsulinmRNA (Villa-Kamaroff et al., 1978). Antigenic determinants for thebacterial p-lactamase and the eukaryotic gene product were detected onfused peptides and on the peptide fragments cleaved from such fusedproteins; however, biological activity of the mammalian components ofsuch immunologically reactive hybrid proteins was not shown.Our approach to the study of mammalian gene expression in bacteriawas to generate a heterogeneous population of clones carrying a DNAsequence that encodes for a selectable mammalian gene product, andthen to select directly those bacteria in the population that phenotypicallyexpress the genetic sequence (Chang et al., 1978). The mammalianenzyme dihydrofolate reductase (DHFR), which catalyzes the conversionof dihydrofolic acid to tetrahydrofolic acid, was especially suitablefor this purpose: the mammalian DHFR has a much lower affinityfor the antimetabolic drug, trimethoprim (Tp) than does the correspondingbacterial enzyme (Burchall and Hitching, 1965). Thus, bacterial' cells that biologically express mammalian DHFR activity are resistant tothe levels of Tp that ordinarily would inhibit their growth. The primaryDNA sequence of plasmids that showed phenotypic expression of themammalian gene product in bacteria could then be analyzed to determinethe specific sequence arrangement that accomplishes expression.Moreover, differences in the level of expression in various clones couldbe correlated with the primary sequence of the clone.198 STANLEY N.COHENFigure 13 summarizes the experimental plan used in these investigations.Partially purified mRNA containing DHFR sequences frommouse cells resistant to the DHFR-inhibiting drug methotrexate (Buellet al., 1978) served as a template for the preparation of double-strandedcDNA using reverse transcriptase and DNA polymerase I. As in thecase of the experiments described above for the ACTH-P-LPH mRNA,homopolymeric dC "tails" were added to the unfractionated cDNA byterminal deoxynucleotidyltransferase and homopolymeric dG tails wereadded to the termini generated by PstI endonuclease cleavage within thep-lactamase of the pBR322 plasmid. Constructed plasmids were introducedinto E. coli by transformation, and plasmid DNA isolated fromTp-resistant colonies was isolated and subjected to fragmentationanalysis by various restriction endonculeases and to DNA sequenceanalysis.As shown in Table 111, the nucleotide sequence in the region of thevector-cDNA junction nearest the 5' end of the DHFR mRNA wasEXTRACT ADD dGdCMAMMALIAN "TAILS"ANDDHFR PLASMIDSYNTHESIZEcDNA COPYDHFR:,"kTPLASMID DNA4ANALYZE SEQUENCE TO DETERMINE HIGH LEVELSTRUCTURE OF "EXPRESSING" CLONES Tp IN MEDIACORRELATE STRUCTURE SELECT FOR ~p~WITH LEVEL OF EXPRESSION BACTERIAL COLONIESFIG. 13. Strategy used to obtain phenotypic expression of a mammalian genetic sequencein Escherichia coli. A heterogeneous population of clones carrying a DNA sequencethat encodes for a mammalian gene product, dihydrofolate reductase (DHFR), thatproduces a selectable trait [high level trimethoprim resistance (TpR)] was generated, andthose bacteria in the population that phenotypically expressed the gene were selected directly.TABLE 111PROPERTIEOSF pDHFR CHIMF.RIPCL ASMIDS"Base pairs (bp) DHFRfrom A of specificATG to center activity MIC Relative ReadingOrientation of 5-bp (unitslmg of Tp DHFR frame ofPlasmid Nucleotide sequence" of cDNA sequence protein) (pglml) activity DHFRAla -13 - I +IpDHFR 7 TG.CA.G.~.GG.GG GGGGGGATGGTT ApDHFR I2pDHRF 13pDHFR 26pDHFR 27pDHFR 28pDHFR 29pDHFR 23" DHFR, dihydrofoIate reductase; MIC, mean inhibitory concentration; Tp, trimethoprim; ND, not detected."he black dots beIow nucleotides indicate homology with the nucleotide sequence at the 3'-OH terminus of 16 S rRNA (i.e., 3'-AUUCCUCCACUAGG-5').200 STANLEY N. COHENcorrelated with other properties of pDHFR chimeric plasmids, includingthe level of DHFR expression. In each instance, bacteria that expressedDHFR activity phenotypicaliy were found to synthesize a protein thathas the enzymic properties, immunological reactivity, and molecularsize of the mouse DHFR (Chang et al., 1978; Erlich et al., 1979).Moreover, the DHFR cDNA segment in such clones was found to be ina different translational reading frame from the bacterial p-lactamasegene into which it had been inserted, suggesting that the biologicallyactive DHFR being produced was not made as part of a fused protein.Together, these findings implied that initiation of translation wasoccurring at the translational start codon (AUG) normally used for thesynthesis of mouse DHFR in its original host. Thus, initiation of a structurallydiscrete and biologically functional eukaryotic peptide wasoccurring in bacteria on a fused (polycistronic) mRNA molecule.One structural feature important in accomplishing such translation"re-starts" is the presence of a ribosomal binding site at an appropriatedistance from the translational start codon; the efficiency ofexpression was found to be strongly influenced by the extent ofhomology of this region of the mRNA with the 3'-OH end of 16 Sribosomal RNA (Shine and Dalgarno, 1974; Steitz and Steege, 1977;Steege, 1977), as well as the distance between the AUG codon and theribosomal binding sequence of the mRNA. The sequence configurationfound to accomplish phenotypic expression of the mouse DHFR geneticsequence in bacterial cells has been used for expression of othereukaryotic proteins in E. coli, and it seems to be generally applicable tothe production of a wide variety of structurally discrete biologicallyfunctional heterospecific proteins in bacterial cells.ACKNOWLEDGMENTSuIt is a pleasure to acknowledge the collaboration of students, postdoctoral researchfellows, and research assistants in my own laboratory, and the collaboration of additionalcolleagues in other laboratories at Stanford and elsewhere in the studies I have reportedhere. Special acknowledgment is due to A. C. Y. Chang for her continuing involvement inmuch of this work. The studies were supported by funds from the National Institutes ofHealth, the American Cancer Society, and the National Science Foundation.REFERENCESAganval, K. L., Buchi, H., Caruthers, M. H., Gupta, N. Khorana, H. G., Kleppe, K.,Kumar, A,, Ohtsuka, E., Rajbhandary, U. L., Van De Sande, J. H., Sgaramella, V.,Weber, H., and Yamada, T. (1970). Nature (London) 277, 27.TRANSPLANTATION OF GENES 201Arber, W. (1965). Annu. Rev. Microbiol. 19, 365.Armstrong, K. A,, Hershfield, V., and Helinski, D. R. (1977). Science 196, 172.Avery, 0. T., Macleod, C. M., and McCarthy, M. (1944). J . Exp. Med. 79, 137.Berg, P., Baltimore, D., Brenner, S., Roblin, R. O., 111, and Singer, M. F. (1975). Proc.Natl. Acad. Sci. U.S.A. 72, 1981.Blattner, F. R., Williams, B. G., Blechl, A. E., Denniston-Thompson, K., Faber, H. E.,Furlong, L., Grunwald, D. J., Kiefer, D. O., Moore, D. D., Schumm, J. W.,Sheldon, E. L., and Smithies, 0. (1977). Science 196, 161.Blobel, A,, and Dobberstein, B. (1975a). J . Cell Biol. 67, 835.Blobel, A., and Dobberstein, B. (1975b). J . Cell Biol. 67, 852. . Bolivar, F., Rodriquez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., andBoyer, H. (1977). Gene 2, 95.Brutlag, D., Fry, K., Nelson, T., and Hung, P. (1977). Cell 10, 509.Buell, G. N., Wickens, M. P., Payvar, F., and Schimke, R. T. (1978). J . Biol. Chem.253, 247 1.Burchall, J. J., and Hitching, G. H. (1965). Mol. Pharmacol. 1, 126.Cabello, F., Timmis, K., and Cohen, S. N. (1976). Nature (London) 259, 285.Chakrabarty, A. M., Friello, D. A., and Bopp, L. H. (1978). Proc. Natl. Acad. Sci.U.S.A.75, 3109.Chang, A. C. Y., and Cohen, S. N. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 1030.Chang, A. C. Y., and Cohen, S. N. (1978). J . Bacteriol. 134, 1141.Chang, A. C. Y., Lansman, R. A,, Clayton, D. A,, and Cohen, S. N. (1975). Cell 6,231.Chang, A. C. Y., Nunberg, J. N., Kaufman, R. J., Erlich, H. A., Schimke, R. T., andCohen, S. N. (1978). Nature (London) 275, 617.Chang, A. C. Y., Cohen, S. N., Nakanishi, S., Inoue, A,, Kita, T., Nakamura, M., andNuma, S. (1979). In "Peptides. Structure and Biological Function. Proceedings ofthe Sixth American Peptide Symposium" (E. Gross and J. Meienhofer, eds.),p. 957. Pierce Chemical Co., Rockford, Illinois.Chang, S., and Cohen, S. N. (1977). Proc. Natl. Acad. Sci. U.S.A . 74, 48 1 1.Cohen, S. N. (1976). Nature (London) 263, 731.Cohen, S. N., and Chang, A. C. Y. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 1293.Cohen, S. N., and Chang, A. C. Y. (1977). J . Bacteriol. 132, 734.Cohen, S. N., and Miller, C. A. (1968). Nature (London) 224, 1273.Cohen, S. N., Chang, A. C. Y., and Hsu, L. (1972). Proc. Natl. Acad. Sci. U.S.A. 69,21 10.- Cohen, S. N., Chang, A. C. Y., Boyer, H. W., and Helling, R. B. (1973). Proc. Natl.Acad. Sci. U.S.A. 70, 3240.Cohen, S. N., Cabello, F., Chang, A. C. Y., and Timmis, K. (1977). In "Tenth MilesInternational Symposium on Recombinant Molecules: Impact on Science and Society(R. F. Beers, Jr., and E. G. Bassett, eds.), p. 91. Raven, New York.Cohen, S. N., Brevet, J., Cabello, F., Chang, A. C. Y., Chou, J., Kopecko, D. J.,Kretschmer, P. J., Nisen, P., and Timmis, K. (1978). In "Microbiology(D. Schlessenger, ed.), p. 217. Am. Soc. Microbiol., Washington, D.C.Cozzarelli, N. R., Melechen, N. E., Jovin, T. M., and Kornberg, A. (1967). Biochem.Biophys. Res. Commun. 28, 578.202 STANLEY N. COHENDanna, K. J., and Nathans, D. (197 1 ). Proc. Natl. Acad. Sci. U.S.A . 68, 29 13.Davis, R. W., and Davidson, N. (1968). Proc. Natl. Acad. Sci. U.S.A. 60, 243.Dawid, 1. B., Brown, D. D., and Reeder, R. H. (1970). J. Mol. Biol. 51, 341.Efstratiadis, A,, Kafatos, F. C., Maxam, A. M., and Maniatis, T. (1976). Cell 7, 279.Erlich, H. A,, Levinson, J. R., Cohen, S. N., and McDevitt, H. 0. (1979). J. Biol.Chem. 254, 12240.Gefter, M. L., Becker, A,, and Hurwitz, J. (1967). Proc. Natl. Acad. Sci. U.S.A. 58,240.Gellert, M. (1967). Pror. Natl. Acad. Sci. U.S.A. 57, 148.Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R.,Hirose, T., Kraszewski, A,, Itakura, K., and Riggs, A. D. (1979). Proc. Narl.Acad. Sci. U.S.A . 76, 106.Grunstein, M., and Hogness, D. S. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 3961.Helling, R. B., Goodman, H. M., and Boyer, H. W. (1974). J. Virol. 14, 1235.Hemnann, R., Neugebauer, K., Schaller, H., and Zentgraf, H. (1978). In "SinglestrandedDNA Phages" (D. T. Denhardt, D. N. Dressier, and D. S. Ray, eds.),p. 473. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Hershfield, V., Boyer, H. W., Yanofsky, C . .Lovett, M. A,, and Helinski, D. R. (1974).Proc. Narl. Acad. Sci. U.S.A. 71, 3455.Heyneker, H. L., Shine, J., Goodman, H. M., Boyer, H. W., Rosenberg, J . , Dickerson,R. E., Narang, S. A,, ltakura, K., Lin, S., and Riggs, A. D. (1976). Nature(London) 263, 748.Hohn, B., and Murray, K. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 3259.Hotchkiss, R. D., and Gabor, M. (1970). Annu. Rev. Genet. 4, 193.Itakura, K., Hirose, T., Crea, R., and Riggs, A. D. (1977). Science 198, 1056.Jackson, D. A. Symons, R. H., and Berg, P. (1972). Proc. Natl. Acad. Sci. U.S.A. 69,2904.Jensen, R. H., Wodzinski, R. J., and Rogoff, M. H. (1971). Biochem. Biophys. Res.Corn~nu~4l3. , 384.Kedes, L. H., Chang, A. C. Y., Housman, D., and Cohen, S. N. (1975). Nature(London) 255, 533.Kelly, T. J., Jr., and Smith, H. 0. (1970). J . Mol. Biol. 51, 393.Keutmann, H. T., Eipper, B. A., and Mains, R. E. (1979). J. Biol. Chem. 254, 9204.Khorana, H. G. (1968). Pure Appl. Chem. 17, 349.Kita, T., Inoue, A., Nakanishi, S., and Numa, S. (1979). Eur. J. Biochem. 93, 213.Kornberg, A. ( 1974). "DNA Synthesis." Freeman, San Francisco, California.Kretschmer, P. J., Chang, A. C. Y., and Cohen, S. N. (1975). J. Bacteriol. 124, 225.Leder, P., Tiemeier, D., and Enquist, L. (1977). Science 196, 175.Li, C. H., and Chung, D. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 1145.Li, C. H., Tan, L., and Chung, D. (1977). Biochem. Biophys. Res. Commun. 77, 1088.Ling, N., Burgus, R., and Guillemin, R. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 3942.Lobban, P. E., and Kaiser, A. D. (1973). J. Mol. Biol. 78, 453.Lovett, M. A., and Helinski, D. R. (1976). J . Bacteriol. 127, 982.Mandel, M., and Higa, A. (1970). J . Mol. Biol. 53, 159.TRANSPLANTATION OF GENES 203Marians, K. J., Wu, R., Stawinski, J., Hozumi, T., and Narang, S. A. (1976). Nature(London) 263, 744.Maxam, A. M., and Gilbert, W. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 560.Meacock, P. A,, and Cohen, S. N. (1979). Mol. Gen. Genet. 174, 135.Mertz, J., and Davis, R. W. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 3370.Meselson, M., and Yuan, R. (1968). Nature (London) 217, 1 1 10.Messing, J., Gronenborn, B., Muller-Hill, B., and Hofschneider, P. H. (1977). Proc.Natl. Acad. Sci. U.S.A. 74, 3642.. Morrow, J. F., Cohen, S. N., Chang, A. C. Y., Boyer, H. W., Goodman, H. M., andHelling, R. B. (1974). Proc. Narl. Acad. Sci. U.S.A. 71, 1743. * Mukai, T., Matsubara, K., and Takagi, Y. (1976). Mol. Gen. Genet. 146, 269.Murray, N. E., and Murray, L. (1974). Narure (London) 251, 476.Nakamura, M., Nakanishi, S., Sueoka, S., Imura, H., and Numa, S. (1978). Eur. J.Biochem. 86, 61.Nakamura, M., Inoue, A., Nakanishi, S., and Numa, S. (1979). FEBS Lett. 105, 357.Nakanishi, S., Kita, T., Taii, S., Imura, H., and Numa, S. (1977). Proc. Narl. Acad. Sci.U.S.A. 74, 3283.Nakanishi, S., Inoue, A,, Kita, T., Numa, S., Chang, A. C. Y., Cohen, S. N., Nunberg,J., and Schimke, R. T. (1978). Proc. Narl. Acad. Sci. U.S.A. 75, 6021.Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., andNuma, S. (1 979). Narrrre (London) 278, 423.Nathans, D., and Danna, K. J. (1972). J. Mol. Biol. 64, 515.Nathans, D., and Smith, H. 0 . (1975). Annu. Rev. Biochem. 4, 273.Olivera, B. M., and Lehman, I. R. (1967). Proc. Narl. Acad. Sci. U.S.A. 57, 1426.Rabbits, T. H. (1976). Narure (London) 260, 221.Rambach, A,, and Tiollais, P. (1974). Proc. Narl. Acad. Sci. U.S.A. 71, 3927.Ratzkin, G., and Carbon, J. (1977). Proc. Narl. Acad. Sci. U.S.A. 74, 487.Roberts, R. J. (1976). Crir. Rev. Biochem. 4, 123.Roberts, R. J. (1977). In "Tenth Miles International Symposium on RecombinantMolecules: Impact on Science and Society (R. F. Beers, Jr., and E. G. Bassett,eds.), p. 21. Raven, New York.Ruogeon, F., Kourilsky, P., and Mach, B. (1975). Nucl. Acids Res. 2, 2365.Scheller, R. H., Dickerson, R. E., Boyer, H. W., Riggs, A. D., and Itakura, K. (1977).Science 196, 177.: Scon, A. P., Ratcliffe, J. G., Rees, L. H., Landon, J., Bennett, H. P. J., Lowry, P. J.,and McMartin, C. (1973). Narure ( L o n d o ~ ~N)e, w Biol. 244, 65.Sgaramella, V. (1972). Pt.oc. Narl. Acad. Sci. U.S.A. 69, 3348.Sgaramella, V.,van de Sande, J. H., and Khorana, H. G. (1970). Proc. Narl. Acad. Sci.U.S.A. 67, 1468.Sgaramella, V., Bursztyn-Pettegrew, H., and Ehrlich, S. D. (1977). In "Tenth MilesInternational Symposium on Recombinant Molecules: Impact on Science and Society(R. F. Beers, Jr., and E. G. Bassett, eds.), p. 57. Raven, New York.Shine, J . , and Dalgarno, L. (1974). Proc. Narl. Acad. Sci. U.S.A. 71, 1342.Silver, R. P., and Cohen, S. N. (1972). J. Bacreriol. 110, 1082.204 STANLEY N. COHENSmith, H. 0.. and Wilcox, K. W. (1970). J. Mol. Biol. 51, 371.Steege, D. A. (1977). Proc. Narl. Acad. Sci. U.S.A. 74, 4163.Steitz, J. A,, and Steege, D. A. (1977). J. Mol. Biol. 114, 545.Streek, R. E., and Hobom, G. (1975). Eur. J. Biochem. 57, 595.Stmhl, K., Cameron, J. R., and Davis, R. W. (1976). Proc. Narl. Acad. Sci. U.S.A. 73,1471.Takano, T., Watanabe, T., and Fukasawa, T. (1968a). Virology 34, 290.Takano, T., Watanabe, T., and Fukasawa, T. (1968b). Biochem. Biophys. Res. Commun.25, 192.Taylor, D. P., and Cohen, S. N. (1979). J. Bacterial. 137, 92.Thomas, M., Cameron, J. R., and Davis, R. W. (1974). Proc. Narl. Acad. Sci. U.S.A.71, 4579.Timmis, K., Cabello, F., and Cohen, S. N. (1974). Proc. Narl. Acad. Sci. U.S.A. 71,4556.Timmis, K., Cabello, F., and Cohen, S. N. (1975). Proc. Narl. Acad. Sci. U.S.A. 72,2242.Timmis, K., Cabello, F., and Cohen, S. N. (1978a). Mol. Gen. Gener. 162, 121.Timmis, K. N., Cabello, F., Andres, I., Nordheim, A,, Burkhardt, H. J., and Cohen, S.N. (1978b). Mol. Gen. Gener. 167, l I.Timmis, K. N., Cohen, S. N., and Cabello, F. C. (1978~). In: "Progress in Molecularand Subcellular Biology" (F. E. Hahn, ed.), Vol. 6, p. 1. Springer-Verlag. Berlinand New York.Vapnek, D., Hautala, J. A,, Jacobson, J. W., Giles, N. H., and Kushner, S. (1977).Proc. Narl. Acad. Sci. U.S.A. 74, 3508.Villa-Kamaroff, L., Efstratiadis, A,, Broome, S., Lomedico, P., Tizard, R., Naber, S.P., Chick, W. L., and Gilbert, W. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 3727.Wade, N. (1974). Science 195, 332.Weiss, B., and Richardson, C. C. (1967). Proc. Narl. Acad. Sci. U3.A . 57, 1021.Wensink, P. C., and Brown, D. D. (1971). J. Mol. Biol. 60, 235.Wensink, P. C., Finnegan, D. J., Donelson, J. E., and Hogness, D. S. (1974). Cell 3,315.Westmoreland, B. C., Szybalski, W., and Ris, H. (1969). Science 163, 1343.Wigler, M., Silverstein, S., Lee, L-S., Pellicer, A,, Ching, Y.-C., and Axel, R. (1977).Cell 11, 223.United States. Patent [l91Cohen et al, I[54] PROCESS FOR PRODUClNG' BIOLOGICALLY FuNCIlONALMOLECULAR CHIMERAS[75] Inventors: Stanley N. Cohea, Portola Valley;Herbert W. Boyer, Mil Valley, bothof Calif.[73] Assignee: Board of Trusteesof the LelandStanford Jr. University, Stanford,Calif.[21] Appl. No.: 1,021[22] ._Filed: Jan. 4,1979. _ . I. .Related US. Application Data[63] Continuation-in-part of Ser. No. 959,288,Nov. 9, 1918,which is a continuation-in-part of Ser. No. 687,430,May 17, 1976, abandoned, which is a continuation-inpartof Ser. No. 520,691, Nov. 4, 1974.[51] Int. C1.3 .............................................. C12P 21/00[52] US. (3. ...................................... 435/68; 435/172;435/23 1; 435/183; 435/3 17; 435/849; 435/820;435/91; 435/207; 260/112.5 S; 260/27R; 435/212[58] Fieldofsearch .............. 195/1, 28 N, 28 R, 112,195178, 79; 435/68, 172, 231, 183~ 6 1 . References CitedU.S. PATENT DOCUMENTSOTHER PUBLICATIONSMorrow et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.3365-3369, Nov. 1972.Morrow et al., Proc. Nat.' Acad. Sci. USA, vol. 71, pp.1743- 1747, May 1974. . .Hershfield et al., Proc. Nat. Acad. Sci. USA, vol. 71,pp. 3455 et seq. (1974).Jackson et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.2904-2909, Oct. 1972.Mertz et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.3370-3374, Nov. 1972.Cohen, et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.1293-1297, May 1973.Cohen et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.3240-3244, Nov. 1973.Cliang et al., Proc. Nat. Acad. Sci, USA, vol. 71, pp.1030--1034, Apr. 1974.Ullrich et al., Science vol. 196, pp. 1313-1319, Jun.1977.Singer et al., Science vol. 18 1, p. 1 1 14 (1 973). .Itakura et al., Science vol. 198, pp. 1056-1063 Dec.1977.Komaroff et al., Proc. Nat. Acad. Sci. USA,vol. 75, pp-3727-373 1, Aug. 1978.Chemical and Engineering News, p. 4, May 30, 1977.Chemical and Engineering News, p. 6; Sep. 11, 1978.Primary Examiner-Alvin E. TanenholtzAttorney. Agent, or Firm-Bertram I. Row landMethod and compositions are provided for replicationand expression of exogenorAs genes in microorganisms.- Plasmids or virus DNA are cleaved to provide hearDNA having ligatable termini to which is inserted agene having complementary termini, to provide a biologicallyfunctional replicon with a desired phenotypicalproperty. The replicon is inserted into a microorganismcell by transformation. Isolation of the transformantsprovides cells for replicatioil and expression ofthe DNA molecules present in the modified plasmid.The method provides a convenient and efficient way tointroduce genetic capability into microorganisms forthe pr*o. d.u-c,. tion of nucleic acids and proteh. such asmedicdly or commercially useful enzymes, which mayhave direct usefulness, or may find expression in theproduction of drugs, such as hormones, antibiotics, orthe like, fmtion of nitrogen, fernentation, utilization ofspecific feedstocks, or the like.14 Claims, No Drawings355IndexAndreopoulos, Spyros, 132Arber, Werner, 45Axline, Stanton G., 23Baltimore, David, 70, 71, 72, 150, 152Beckwith, Jonathan, 102, 103, 128, 169Belasco, Joel, 174Berg, Paul, 47, 58, 88, 91, 167Berg et al. letter, 73, 76, 84, 89, 94, 106, 120, 125, 128, 129, 135, 137COGENE membership, 122dA-T joining, 40, 69Gene splicing versus recombinant DNA, 42National Academy of Sciences Committee, 55, 69, 70, 82, 87Opposition to patent, 155, 158, 160, 164Recruiting Cohen to Stanford, 17, 159Relationship to Cohen's work, 43Relationship to Lobban's work, 41, 43, 164Role in legislation, 98, 102, 105Role in Stanford Biosafety Committee, 135Bibb, Mervyn, 172, 180, 183Biosafety Committee at Stanford, 135Botstein, David, 69, 144, 171Boyer, Herbert, 40, 66, 68, 82, 92, 93, 105, 112, 114, 117, 133Awards, 12, 40Berg et al. Committee & letter, 70, 73, 106Cohen-Boyer patent, 147, 151, 158, 160, 163, 164, 165, 167EcoRI work, 47First Cohen-Boyer experiments, 45, 50First Cohen-Boyer paper, 55, 56Genentech, 60, 98, 105, 112, 151, 154, 155, 157, 158, 167Gordon conference, 42, 56, 58, 69, 129, 164Honolulu, Hawaii meeting on plasmid biology, 46, 48Nature of Recombinant DNA experiments, 119, 128Recombinant DNA successes, 52, 95, 99Xenopus work, 62, 69Breedis, Charles, 8Brenner, Sydney, 82, 84, 129Brown, Donald, 58, 59, 69, 108, 109Burroughs Wellcome Fund Award, 22, 35, 113Cabello, Felipe, 166Cape, Ron, 111Casadaban, Malcolm, 169Cavalli-Sforza, Luca, 25, 144Cetus, 105, 113, 126, 153, 155Chakrabarty, Al, 75Chang, Annie, 30, 58, 62, 67, 75, 151, 157Chang, Shing, 117, 184Chargaff, Erwin, 102, 105, 114, 116, 128, 136237Chi1776, 84, 109, 125Biological containment, 85, 98Clayton, David, 62, 140Clowes, Roy, 30, 74, 78, 138COGENE (Committee on Genetic Experimentation), 121, 134Cohen, Joan, 16, 89, 142, 198Cosloy, Sharon, 35Curtiss, Roy, 74, 78, 84, 93, 95, 125Davidson, Norman, 43, 46, 50, 55, 57, 59, 170Davis, Ronald, 47, 65, 67, 71, 129, 160, 164Department of Genetics, 21, 24, 25, 46, 69, 143, 159, 165Cohen as Chair, 21, 165Cohen's appointment to, 22, 24, 143, 194Division of Clinical Pharmacology, 22, 24, 97, 113, 143Duke University, 17Housestaff training, 12Ehrlich, Henry, 112, 184Falkow, Stanley, 16, 26, 31, 32, 49, 51, 70, 78, 112, 170Collaborations, 95Concerns, 81Discussion in Honolulu, 48, 151Plasmid Nomenclature Working Group (Plasmid Committee), 74, 76, 93R factors, 27Federman, Daniel D., 24, 143Fildes, Bob, 112Filter Affinity Transfer, 184Fredrickson, Donald, 117, 118, 119, 141Friends of the Earth, 124Gellert, Martin, 37, 41Gilbert, David, 179, 189Gilbert, Walter, 59, 60, 98, 112Glaser, Donald, 111Goodman, Howard, 46, 47, 55, 62, 158Handler, Phillip, 69, 71Hedgepeth, Joseph, 46, 48, 160Helinski, Donald, 29, 46, 74, 93, 94, 95Helling, Bob, 42, 52, 55, 61, 151, 157, 158Herzenberg, Leonard, 145Higa, Akiko, 35, 41Hogness, David, 19, 34, 60, 67, 70, 82, 93, 105, 166Holman, Halstead (Hal), 24, 107, 125, 128Recruiting Cohen to Stanford, 18, 19Role in Legislation, 121Support for Cohen at Stanford, 22, 29U.S. Senate Hearing, 109Hopwood, David, 142, 169, 173, 180, 182, 183, 195238Horowitz, Larry, 106, 109, 119, 123Hsu, Leslie, 35, 36, 41, 42, 52Hurwitz, Jerard, 14, 15, 17, 20, 30, 37, 41, 69, 175, 181Interferon, 146Jackson, David, 38, 39, 43, 69, 131, 164John Innes InstituteSabbatical leave at, 142Kaiser, Dale, 17, 34, 39, 41, 43, 64, 66, 131, 159, 164Keene, Senator Barry, 123, 127Kennedy, Donald, 147, 163, 165Kennedy, Senator Edward, 106, 109, 118, 119, 120, 121, 123Khorana, H. Gobind, 37, 38, 41, 47, 48King, Jonathan, 102, 103Kopecko, Dennis, 170Kornberg, Arthur, 17, 19, 20, 37, 65, 132, 148, 155, 158Lappé, Marc, 125, 128Lederberg, Joshua, 24, 46, 47, 134, 142, 157, 159, 164, 172and Sgaramella, 47As chairman of Genetics Department, 24, 144, 168, 194At Asilomar, 83, 85, 91Cetus, 111Discovery of Transduction, 41Early work, 27, 41Recombinant DNA Guidelines, 83, 85Research interests, 25Role in legislation, 118Submission of Xenopus DNA cloning paper, 69, 72SUMEX-AIM, 25Lehman, Robert, 37Lin-Chao, Sue, 175, 178Lobban, Peter, 19, 34, 35, 38dA-T tailing, 43, 66, 131, 164Relationship to Cohen's work, 43Thesis dissertation, 53Mandel, Morton, 35, 41Massey, William, 140, 156McClintock, Barbara, 82, 130, 171McDevitt, Hugh, 18, 184McElheny, Victor, 72, 73, 150Meacock, Peter, 169, 177Medawar, Peter, 8MEDIPHOR, 23Melmon, Kenneth, 21, 147Merigan, Thomas, 19, 23Mertz, Janet, 41, 47, 65, 129, 160, 164Michaels, Alan, 148239Miller, Christine, 21, 30, 178, 180Morrow, John, 108, 158Dispute over patent, 151EcoRI findings, 47Xenopus work, 59, 65MYCIN, 23, 25Nakanishi, Shigitada, 63Nathans, Daniel, 47, 71National Academy of Sciences Committee, 55, 71, 82, 87National Institute of Health (NIH), 16, 25, 92, 117, 149, 152Chairpersons of, 145Cohen's early career at, 11, 13, 18, 22, 37, 144Funding, 23, 29, 92, 96, 100, 145, 146, 161, 188Guidelines, 77, 78, 87, 99, 108, 110, 120, 122, 137, 141, 168Patent agreement, 162New York Times, 14, 72, 90, 150Nordstrom, Kurt, 178Novick, Richard, 14, 31, 74, 78, 80, 87, 119, 128, 177Numa, Shosaka, 63Oishi, M., 35Plasmid (origin of term), 25Plasmid Nomenclature Working Group (Plasmid Committee)Role in Asilomar meeting, 87, 92, 94Role in plasmid nomenclature, 76, 123, 138Pollack, Robert, 42, 69, 73pSC101 plasmid, 57, 58, 108, 163, 169, 178Distribution of, 68, 69, 71, 88, 138Origin of, 75, 165Use as vector for DNA cloning, 51, 53, 60, 95Recombinant DNA Advisory Committee (RAC), 81, 86, 94, 96, 100, 105, 138, 141Reimers, Niels, 147, 150, 154, 155, 158, 160, 163Robertson, Channing, 148Robinson, William, 20, 56, 168Roblin, Richard, 71, 88Rogers, Paul (Congressman), 100Rosenzweig, Robert (Bob), 98, 101, 138, 153Rowland, Bert, 150, 151, 156, 157, 160, 161Rownd, Robert (Bob), 15, 26, 27, 28, 31, 178Rutgers University, 5Sambrook, Joseph, 49Schimke, Robert, 60, 64, 97, 167Sgaramella, Vittorio, 160Cohesive ends generated by Eco RI, 47, 65, 160, 164DNA joining, 38, 41Shapiro, Lucy, 12Sharp, Phillip, 43, 46, 49, 50, 59, 170240Shooter, Eric, 145Shortliffe, E. H. (Ted), 23, 25, 31Shottel, Janet, 173, 180Sierra Club, 124Singer, MaxineSinger- Söll letter, 55, 67, 68, 69, 129Sinsheimer, Robert (Bob), 102, 104, 128Söll, DieterSinger- Söll letter, 55, 67, 68, 69, 129Staggers, Harley (Congressman), 101Staphylococcal DNA cloning, 15, 57, 58, 61, 67, 69, 78Stark, George, 185Stead, Eugene, 12Stockdale, Frank, 19Sugden, William, 49, 55SUMEX-AIM, 25SV40 DNA, 43, 47, 58, 69, 70, 73, 82, 131Swanson, Bob, 152, 154, 155Ten Hagen, Kelly, 179Timmis, Kenneth, 166University of Pennsylvania, 9, 14, 16, 101Vector, 129von Gabain, Alex, 174Wald, George, 102, 103, 128Watanabe, Tsutomu, 27, 31, 46, 92Watson, James, 14, 71, 100, 104, 120, 132, 134At Asilomar, 91Weissman, Sherman, 71Wilderness Society, 124Xenopus DNA cloning experiments, 44, 56, 64, 65, 66, 69, 72, 140, 157, 166Xu, Feng-Feng, 175Yanofsky, Charles, 148Yielding, K. Lemone, 10, 37Ziff, EdwardZiff article, 163Zimmerman, Burke, 105, 123Zinder, Norton, 41, 71, 100