reese mee , HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE GRANT APPLICATION _HyPE PROGRAM NUMBER Rol C4 1L&%C-ol FORMERLY —— REVIEW GROUP COUNCIL [Month, Year! DATE RECEIVED TO BE COMPLETED BY PRINCIPAL INVESTIGATOR [items 1 through 7 and 1SA) _yhy TITLE OF PROPOSAL (Do not exceed §3 typewriter spaces} Genetics of Bacteria 2, PRINCIPAL INVESTIGATOR T3, DATES OF ENTIRE PROPOSED PROJECT PERIOD (This anolicotioa) 2A, NAME (Last, First, Initiat) FROM THROUGH Lederberg, Joshua 15 May 1974 7 15 May 1977 4, TOTAL DIRECT COSTS RE- |S. DIRECT COSTS REQUESTED 2B, TITLE OF POSITION Professor of Genetics ipa FOR PERIOO IN FOR FIAST 12-MONTH PERIOD ATEM3 $195 ,000 . ,$ 60,000 IG. MAILING ADDRESS (Street, City, State, Zip Code) Department of Genetics Stanford University School of Medicine Stanford, California 94305 . &. PERFOAMANCE Silcis) (See /astructions) Department of Genetics Stanford University School of fMedicine Stanford, California 94305 30, DEGREE 2E, SOCIALSECURITYNO. 2F.TELE- Area Coda TELEPHONE NUMBER AND NSION on | 415 497-5801 2G, DEPARTMENT, SERVICE, LABORATORY OR EQUIVALENT {See Instructions} Department of Genetics .2H,. MAJOR SUBDIVISION (See instructions) School of Medicine ¥, Research Involving Human Subjects (See fastructions} A.A NO B.D YES Approved: c. (7 YES — Pending Reviews Dato Congressional District No. 17 8. iaventions (Renewal Applicants Only - See [nstructions) . A.KHNO B.() YES — Not previously reported . CCLIYES — Previously reportea -. TO BE COMPLETED BY RESPONSIBLE ADMINISTRATIVE AUTHORITY (items 8 through 12 ang 158) 9, APPLICANT ORGANIZATION(S) (See lnsiructions) Stanford University Stanford, California 94305 TRS No. 94-1156365 . Congressional District No. 17 Tt. TYPE OF OAGANIZATION (Checx applicable item} Clreperal Cistate CJ LOCAL XX OTHER (Specify) Private non-profit university NAME, TITLE, ADDRESS, ANO TELEPHONE NUMJ3ER OF OFFICIAL IN BUSINESS OFFICE WHO SHOULD ALSO 8€ NOTIFIED 1f AN AWARD IS MADE 42, K. D. Creighton Deputy Vice President for Business and Finance . Stanford University, Stanford, Californii 10. NAME, TITLE, AND TE LEPHONE NUMBER OF OFFICIALIS) SIGNING FOR APPLICANT ORGANIZATION (S) Revart D. Siamaas. Contracts and Grants Manager c/O ‘Sponsored Projects. Office. ~ (415) 497 2883 . c+ ~~ Tetephone Number (s} 415) 497 2251 © 94305 Telephone Number — 13, IOENTI HGANIZATIONAL COMPONENT 10 RECEIVE CRED FOR INSTITUTIONAL GRANT PURPOSES (Sce tastructions) 01 School of Medicine. 14, PHS ACCOUNT NUMGER [Enter ieknown] 458210 15. CERTIFICATION AND ACCEPTANCE, We, the undersigned, certify that the statements horein are truce and com knowledge and accept, as to any grant awarded, the obligation to comply with Public Health Serv award, Me plete to the best of our ice terms and conditions in effect at the time of the SIGNATURES (Signatures required on A. SIGNATURE OF PERSON NAMED IN ITEM 2A OATE . 25 April 1974 original capy only. Use ink, ’Por™ signatures not acceptable} OATE PHS-398 Rev, 3-70 5. SIGNAT URS IS) OF PERSONISTNAMED INJTEW 10” Rod sit 1D Sf Yana Sf | DETAILED BUDGET For FIRST 12-4 .TH PERIOD! cy, ees : ST POR FINST W2-8. TH PERIOD) 5 715/74 5/15/69 1, PERSONNEL (List all personnel cndaged on project) pmeee AMOUNT REQUESTED Omit cents) NAME (Last, lirst, initial) TITLE OF POSITION uns. TOTAL Lederberg, Joshua _ Principal Investigator or 15% Program Director Ehrlich, Stanislav Research Associate | 752 Elkana, Yehudit Sr. Res. Assistant {100% Bursztyn, Hela Sr. Res. Assistant | 75% Evans, Peter ; Lab. Technician 50% Jennings, Johnnye ~ Lab. Technician 754% Secretary Secretary - 20% — i : . . TOTAL ——-—_——_-__» | 5 45,689 2, CONSULTANT COSTS (Include Fees and Travel $s 3, EQUIPMENT (Itemize) s 4,000* 4 SUPPLIES Research chemicals, laboratory glassware $s 8,000 "STAFF @. DOMESTIC s 1,000 TRAVEL (See Instructions) b. FOREIGN $ 6. PATIENT COSTS (Separate Inpatient and Outpatient) . : $ 7. ALTERATIONS AND RENOVATIONS ‘ . — $s 8. OTHER EXPENSES (Itemize per instructions) Reprints and publication costs, reference materials, Equipment maintenance, communications s 1,311 9. Subtotol — Items Pthry 8 => |s 60,000 10. TRAINEE EXPENSES See Instructions) PREDOCTORAL No. Proposed 5 FOR a. STIPENDS | POSTDOCTORAL No. Proposed s 7 OTHER (Specify) No. Proposed s TRAINING DEPENDENCY ALLOWANCE, $s GRANTS ‘ ; TOTAL §TIPENC EXPENSES —————_—__» [5 b. TUITION ANO FEES ONLY ¢. TRAINEE TRAVEL (Describe) Subtotol ~ Troinec Expenses 12, TOTAL DIRECT COST (Add Subtotats, Itenis 9 and 11, and enteron Page 1) 60 ,000 Substitute Budget Page 5-72 For Forms PHS 398 and PHS 2499-1 GPO 950.751 Supplement ary I.ormation for Personnel Costs®. .equesied - Its the policy of the DHEW to omit specific salary rates or amounts for individuals from those copies of grant applications which are made available to reviewing *” ‘consultants. Therefore, a substitute budget page is enclosed and must be used in lieu of the present budget page in all competing applications. Note that individual salaries will not be shown on the substitute budget page; however, if a grant is to be awarded as a result of this application, the awarding unit will need information concerning specific salaries. Please use the space below to list all personnel shown on the substitute budget page, and include the salary and fringe benefits requested for each, following the appropriate guidelines in the Information and Instructions. THIS INFORMATION WILL BE USED ONLY BY PHS ADMINISTRATI VE STAPF. DE SCRIPTION {liemize} " AMOUNT REQUESTED (Omit cents) TINE OR PERSONNEL EFFORT FRINGE NAME TITLE OF POSITION mans. | SALARY BENEFITS TOTAL Lederberg, Joshua PRINCIPAL INVESTIGATOR _ 15% |-- a S$ o Ehrlich, Stanislav Research Associate 75% | 9,000 1 530 10 ,530 Elkana, Yehudi-c Sr. Res. Assistant 100% | 9,000 1,530 10 ,530 Bursztyn, Hela _ Sr. Res. Assistant 75% | 8,625 ‘1,466 10,091 Evans, Peter Lab. Technician 50% | 4,500 766 5,266 Jennings, Johnnye Lab. Technician 75% | 5,925 1,007 6 ,932 Secretary Secretary 20% | 2,000 340 2,340 39,050 6 ,639 45 ,689 TOTAL - Same as total for personnel on v5 Substitute Detailed Budget Page ‘ 45 ,689 , DATE OF DHEW AGREEMENT: - CO WAIVED . ie aa — % SBAV" ~ (CU UNDER NEGOTIATION WITH: (See tnstruction ] «, NIDC June 26, 1973 a LD “IF THIS IS A SPECIAL RATE (e.a. off-site], SO INDICATE. -— Supplesentery Inforastion for Personnel Costs 5-72 For Forms PRS 338 and FHS 2499-1 GPO 939-790 SEC riON Th — PRIVILEGE GI CU UN Puen TIM tY BUDGET ESTIMATES ror€ . YEARS OF SUFPORT REQUESTED FRE IUBLIC HEALTH SERVICE DIRECT COSTS OfLY (Omit Cents) . 3ST PERIOD ADDITIONAL YEARS SUPPORT REQUESTED (This application only) DESCRIPTION (SANE AS OE- > - - TAILED BUDGET! | 2NOD YEAR 3RD YEAR 4TH YEAR 5TH YEAR | 6TH YEAR ITH YEAR PERSONNEL costs B45 ,689 48,384 |$51,233 CONSULTANT COSTS (include fees, travel, etc.) EQUIPMENT + 4,000 4,500 4,500 SUPPLIES . 8,000 9,000 10 ,000 DOMESTIC TRAVEL 1,000 1,000 1,000 FOREIGN PATIENT COSTS ALTERATIONS AND RENOVATIONS OTHER EXPENSES 1,311 2,116 3,267 TOTAL DIRECT COSTS $60,000 $65,000 [$70,000 TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1, Item 4) —————» | S$ 495 »000 ed may not be obvious. For future years, justify equipment costs, as well as any REMARKS: Justify all costs for the first year for which the ne f ; ; { increase in personnel costs is requested, give percentage, (Use continuation significant increases in any other category. if a recurring annua page if neede..} * Equipment: The budgeted amount of $4,000 is based on past experience for requirements for replacement and updating of centrifuges, spectrophotometers and other laboratory instrumentation, as well as for autoclaves and other laboratory hardware. We are now reviewing a number of items that are approaching obsolescence or may require major repairs (e.g. a $3,000 item on a leaky, large autoclave, and portions of a distilled water system), and are working out strategies for coping with these requirements - in some cases, too pressing to tolerate a 9 month delay. We will therefore offer detailed justification for specific items at a later date during the review of this application, and in any event will of course require specific approval from NIH for major purchases. PHS-398 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page A. INTRODUCTION 1. Objective: The central research objective of this laboratory for many years has been the understanding of genetic mechanismsin bacteria. For the last decade, we have put aside studies on conjugal exchange in Escherichia coli and phage-mediated transduction in Salmonella (Lederberg, 195867) in favor of the DNA~mediated transfer of genetic information in Bacillus subtilis. Our early studies were among the first to be concerned with linkage and with the details of DNA integration in this system. More recently, and up to the present time, our efforts are directed at the gamble of finding ways to achieve molecular translocation, that is to introduce genetic information, according to the free choice of the experimenter, into the genome of say B. subtilis. Despite the epochal advances in technical facility and theoretical understanding that many workers have achieved with respect to the replication of DNA, and to its integration in the process of genetic trans- formation in a variety of bacteria, genetic exchange has still been restricted to pairs of rather closely related species. It would be of great theoretical and practical importance to be able to introduce, for example, sequences of synthetic polynucleotides or of mammal*.an DNA into a bacterium or a virus genome. However, the transforming systems that have been described to date all show a high degree of specificity; evidently a critical step in the integration of an entering fragment of DNA is the probative formation of a heteroduplex between the chromosome and the fragment, and the rejection of unmatchable, or poorly matched, pairs. Our previous efforts to surmount this obstacle will be summarized below; ‘they have been superseded by Sgaramella's discovery ( %Y, elaborated here, that the polynucleotide ligase coded by phage T4 (T4-ligase) grown in E. coli is capable of mediating the terminal joining of two DNA duplexes in addition to the already known function of sealing nicks within a well formed duplex. With certain tricks the sealing function can also be exploited for the purpose of achieving molecular translocations, as has been done in parallel studies by Paul Berg and his associates €-%3_).(3¢/ Our specific objective is the further exploration of the chemical mechanism and biological effect of terminal joining of biologically specific and active DNA molecules, the correlation of chemical and biological linkage, the facili- tation of preparing DNA from various sources to allow such joining - in short, THE DEVELOPMENT OF MOLECULAR TRANSLOCATION AS A ROUTINE METHOD OF STUDY OF CELLULAR GENETICS. There is hardly a problem in genetics, pure or applied, that might not be influenced by the technical ability to study the function of a DNA segment in a well standardized context of a bacterial or a viral genome. However, we do not underestimate the difficulties of reaching that goal, and specific applied problems that we hope to attack in this way are under the heading "D. Signifi- cance" rather than as realistic expectations for the immediate period of this prospective grant. It is to be expected, and certainly not to be discouraged, that many other investigators will capitalize on these applications, as has been the case, of course, for such discoveries as conjugation, transduction and lambda-lysogeny. PHS-398 Page Rev. 2-69 . GPO : 1968 © - 350-360 Continuation page DO NOT TYPE IN THIS SPACE-BINDING MARGIN 2. Background: The general features of transformation are well known, and discussed in much detail in numerous reviews and texts (e.g., Stent ( of) Watson ( SH)» Hayes ¢26 ). A review by Hotchkiss and Gabor (337 ) cites 241 references through 1969, almost all of them relevant to the present discus- sion. We will then not undertake a monographic survey or complete bibliography here. The specificity of transformation, e.g. the rejection of E. coli DNA by B. subtilis, pervades the literature. However, few published reports are directed at pushing the empirical upper limits, perhaps < 1079, on the relative efficiency of gene transfer between these species, or sinilar situations. Un- doubtedly, we have followed a common procedure in not bothering to publish such negative results in detail. Gene transfer between B. subtilis and other B. spp. is ig greatly hindered, but nevertheless does occur (cf. Wilson and Young, 1972 ( 48), whose results concur with our own, R. Harris, unpubl.). The behavior Fe)» whe is consis- tent with the model of a requirement for regular duplex formation in local segments mentioned above (in contrast, for example, with a restriction (and host- nodification}bysten as is reported for Hemophilus (Smith and Wilcox, 1970 GA. Hotchkiss ( “773) has discussed the relative exclusion of some markers arising by mutation possibly involving heterogeneity at a single base pair. These findings help to delineate, rather than to solve, the problem of achieving molecular translocation involving arbitrary, alien sequences. An overview of mutation in bacteria compared to eukaryotes also suggests a basic difference in the role of chromosome inversion and translocation. These processes dominate variety and species formation in eukaryotes; they occur only exceptionally in bacteria, and then perhaps mainly in relation to the integra- tion and de-integration of episomes. For this reason, in previous grant applications, I had postulated that translocation-mediating enzymes were a later evolution, connected with mechanisms of gene-regulation more complex than the sequential expression of markers in a linear (or circular, or simple multi-segmented) pattern - the concept associated with the operon model of gene regulation in prokaryotes. (One could also argue that eukaryote chromosomes have evolved multiple recognition sites, i.e., that this is one role of "redundant-sequence DNA "across which translocations could be sealed without invoking new enzymes. The report of t ane ie between mouse and human chromosomes in somatic cell hybrids ( Ruedl le G2 (#f) jo tends to argue against such a role). For this reason, some of our previous efforts were directed at the examination of DNA-repair mechanisms of a eukaryote in vivo, that is in eggs of Xenopus injected with bacterial DNA. These studies, reported further in "4. Progress Report", have, however, been overtaken by Sgaramella's findings with T4-ligase. This recent paper, which is appended, is the main foundation of our intended further work. Previous work had indicated terminal joining of two synthetic polynucleotides (Sgaramella et al., 1970 197%, each of which had based-paired ends (a deoxynucleoside 5' phosphate paired with a complementary 3'hydroxyl), a condition of the ends that may be termed "flush". The present paper takes advantage of the known condition of phage P22 DNA as a flush clipped ensemble of segments which are tetminally redundant, but produced as if by a random cut in a circular molecule. Therefore, almost every end is different, and the ease PHS-398 Page Rev. 2-69 . ‘ GPO : 1969 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page of terminal joining then argues against sequence-specificities for the ligase. Furthermore, P22 did not join terminally to linearized SV-40 DNA, the latter having thus been shown to have not a clipped but a cohesive end, i.e., an over- lapping simplex that would seek its complement on another strand. This SV-40 DNA (obtained by the action of a sequence-specific nuclease restriction enzyme on circular SV-40) could, however, be homo-oligomerized either with T4 or E. coli ligase, both enzymes being able to seal a contrived, doubly nicked duplex. The paper itself gives a more complete, and possibly clearer, account. The problem of molecular translocation can then be reduced to that of securing flush ends on biologically interesting DNA. Alternatively, various restriction enzymes might have just the appropriate specificity to generate useful cohesive ends, or terminal polymerases may be used to add synthetic homo-polymer cohesive ends to existing DNA molecules, an approach alm contemplated in the previously submitted applications in this series. Berg's group will be emphasizing the second and third of these approaches, and have, of course, al~ ready achieved an outstanding result with SV-40 and lambda; we will be concen- trating on terminal joining. In order to pursue the biological activity of the oligomers, we have developed a transfection system (see Progress Report) for P22 DNA. (In the light of unforeseeable hazards with derivatives of SV-40, we prefer not to pur- sue work with this as an animal virus in tissue culture). Although most promis- ing, the transfection system needs further improvements (which we foresee should be possible) before we can efficiently test the oligomers for biological activity. 3. Rationale: This is difficult to state differently from the operational and situational (historical) aspects of our proposal. The ultimate rationale is the DNA theory of heredity, namely that the information encoded in base- sequences of DNA molecules is the material basis of heredity. Hence, manipula- tions by chemical, physical and enzymatic means of DNA molecules afford a way of understanding and controlling the hereditary processes of cells. The particular cells we are concerned with are bacteria (and their viruses); and the specific manipulation that now gives us new opportunities for genetic experimentation is the end-to-end joining of DNA segments, and eventually the insertion of new genes into established genomes. 4. Comprehensive Progress Report: a. Period: September 1968 to December 1972. b. Summary: (see also A.2.Background) We tried various approaches to the chemical cross-linking between DNA molecules, the addition of synthetic cohesive ends, and laying the basis for identifying a terminal-joining enzyme in frog eggs. These false starts were superseded by the identification of terminal-joining activity in T4-ligase and its use in the formation of oligomers of P22 DNA (%), at , We have also searched for deletion mutations in B. subtilis and were surprised to find that they are very rare, if they occur at all in our strain. Heterospecific tzansformations (B. subtilis x B. globigii) were studied and PHS-398 Page Rev. 2-69 $ . GPO : 1989 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page the model that sequence hemology is required for efficient transformation was supported, We have made a detailed analysis of the effects of chlorine on DNA, but eventually concluded that its effects on DNA breakage were incidental to effects on proteins, and that the latter was the principal site of cell damage. How- ever, chlorine is a (feeble) mutagen, and has an enigmatic effect on the burst size as well as viability of treated phage (lambda). It therefore deserves further study as a possible environmental pollutant of biological consequence. A variety of other incidental findings are indicated in the list of pub- lished titles. c. Detailed Report: The most salient findings have already been outlined under "Background" and in the Sgaramella paper (1972 , @— ), which should be regarded as part of this application. ZA « A range of other studies avowedly of lesser importance is detailed in the remaining list of publications (see d.). Work not yet published includes: 4. (R. Harris). A study of the specificity of a nuclease produced by B. globigii and not by B. subtilis. This nuclease attacks B. subtilis DNA more rapidly than that from B. globigii and may therefore resemble "restric- tion enzymes" in specificity. The DNA from different hybrids is being examined to look into the role and nature of a host-modification system. However, there is no evidence of such an enzyme with differential specificity in the competent strain of B. subtilis as might be hypothesized to account for the specificity of DNA in transformation. ” ii. (1. Majerfeld). The cross-linking of DNA with various agents, of which glyoxal and glutaraldehyde appear to be the most promising. (The rationale was that chemically linked DNA might be copied with DNA polymerase with a.mere skip across the link in the template. However, material isolated so far has not been well enough defined for a cogent test of the concept). The work was interrupted by Mrs. Majerfeld's emigration to England with her husband prior to the completion of her Ph.D. research. She may, however, resume it at Sussex. iii. (B. Brandt and J. Wachtel, in press). On the hypothesis that eukaryotes possessed an enzyme for molecular translocation, we tried to develop a system for studying the effect of frog egg enzymes, in vivo, on . injected bacterial DNA. Following Gurdot!*DNA synthesis was demonstrable rol on the injected templates. However, it proved to be too difficult to recover workable amounts of "repaired" DNA from injected eggs, and attemtion was then directed to the enzymes in the extracts. These have been shown to contain a DNA-polymerase with variations in template specificity similar to those reported for the intact eggs by Gurdon. That is, Xenopus laevis DNA, undenatured (but treated with DNAse I) was a preferred primer, compared to E. coli DNA or to poly d(A,T). E. coli polymerase I prefers denatured DNA from various sources. Larvae and immature ovaries yielded enzymatic activities with still different patterns of preference. However, on 50-fold purification, the egg extract en- zyme was less discriminating, perhaps owing to the removal of a DNAse-inhibitor. PHS-398 Page : Rev. 2-69 GPO : 1969 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page A ligase has also been demonstrated in these extracts, but has not yet been characterized, i.e. for temrinal joining activity. iv. (V. Sgaramella and H. Bursztyn, manuscript in preparation). Transfection in P22. The more or less fortuitous amenability of P22 DNA to terminal joining by T4 ligase naturally led to an inquiry on the biological activity of the oligomers. Published work in transfection with P22 DNA has been remarkably discouraging, efficiencies about 10-9 having been reported with spheroplasted hosts (1%) Sin order to attempt correlative transduc- tion, we would prefer intact bacteria. Attempts to condition Salmonella by cold shock and CaCl2 which has given spectacular results with E. coli (Hg <7 were unrewarding. However, supernates of shocked E. coli cells were found to condition an R strain of S. typhimurium LT2 to a low rate of transfective com- petence. (The supernate factor is, of course, under close study). Subsequently it was found that limited treatment of P22 DNA with exonuclease would further augment the efficiency of transfection to a level now in the range of 1077 to 10-8, We have not evidently exhausted the possibility of further improvement which would furnish another valuable tool. These rates are still too low to expect the transduction of bacterial markers to be observed - nor have we done so as yet. PHS-398 ‘ Page Rev. 2-69 GPO : 1968 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page B. SPECIFIC AIMS We have now passed the probing stage, and with the T4 ligase intend a systematic study of ways to achieve molecular translocation. Perhaps this is also the place to remark about my personal involvement in this research. During the past five years, I have undertaken heavy commit- ments in pursuing the social and ethical aspects of science as a matter of self-education and in relation to national policy (e.g. as a consultant to the ACDA on the Biological Warfare Treaty negotiations, and as a member of the National Advisory Mental Health Council); and in public education and under- standing of science as a weekly columnist for the Washington Post, and as an occasional witness before Congressional committees. Such activities, however one evaluates then, inevitably compete with lab- oratory research and I could hardly point to this period as one of notable productivity in molecular genetics. For almost a year, however, I have concluded that I had now made my main contribution to public service and could concentrate again on my laboratory responsibilities - and this has been greatly reinforced by the work on the T4 ligase summarized herein. While I still must own to heavy administrative and some public responsibilities, and am stila involved in other res ch projects unrelated to this (involving computer intelligence and participation in the Mars 1975 Viking mission) I am fortunate that, for vattous reasons, these also promise to be less demanding. And, of course, the graceful termination of my regular column writing is a particular bonus from this standpoint. I should stress that the present grant is the only substantial support to which I can look for laboratory work in molecular genetics - the rest being limited to sharing in the department's training grant for stipends for graduate students and fellows. My name is associated with these and other funds in an administrative more than a personal capacity, and I do not have recourse to them for this line of research. C. METHODS OF PROCEDURE The detailed methodology is outlined.in attached and referenced papers, and this section will address a strategic outline. RES EMRE PLAN - OUTLINE Work in progress sets a substantial momentum. Our plans are a combination ‘of opportunistic forays from recent discoveries and systematic exploration of plausible alternatives. In relation to the overall goal of molecular trans- location we perceive the following set of interrelated issues: (1) How generally to achieve proper flush clipping of DNA; - (11). The choice of donor DNA; ‘ , (111) The choice of co-donor and of recépient genomes and the associated biological assay systems. We have also to consider (iv) The functions of terminal joining in phage and by hypothesis in eukaryotes and further study of ligase specificities; (v) Other problems that may attend the integration ard functioning of ingerted poauences, and ~ — PHS698 . Page Rev. 2-69 GPO : 1969 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN a“ , Continuation page 7 ne ( (vi) Specific problem-oriented applications of these new methods. (i) FLUSH CLIPPING DNA. The availability of monomer P22 DNA of standard size facilitates the assay for flush clipping of other DNA's of different length or composition. If these are differentially labeled, terminal joining will give stable covalent complexes that can be separated in the centrifuge. We plan then to explore the flush clipping of various phage and bacterial DNA's by various enzymes and physical treatments, and also followed by exo- nuclease or repair-polymerase intended to rectify the ends. We do know that simple sonication does not work. Some evidence that ravelled ends bind the enzyme needs to be elaborated: if ravelled ends do compete we need to find ways to purify the flush clipped component of mixtures. Some endonucleases that reputedly flush clip may leave short simplex ends that could, however, be rectified by exonuclease or polymerase. _. (44) DONOR DNA. P22 and FRAGMENTS, The P22-P22 (homo) oligomers may give. the first opportunity to examine a biological correlate of terminal joining - .1if they are biologically active. Obviously, testing-these for infectivity or for rescuable markers is high on our agenda. If this succeeds much interesting work remains on the further dissection of the fragments. It also opens the opportunity to look at the addition of torn fragments of P22 (with one flush end) to intact P22 with respect to the genetic information that may then be introduced from marked phage genotypes. pt Re E. coli episomes offer a wide range of DNA sequences with predictable biological activity - for example, the tryptophan sythetase of 80 (E. coli). -/ If inserted into a continuous subtilis sequence the complex might transform B. subtilis auxotrophs to give an easily recognizable alien gene product. As always, the proper clipping pf these inputs i crucial . Defective bacterial phages of B. subtilis, reported bitte Bebe te arek OSrF sh ends 1X54 , might afford ready-made fragments for temrinal joining to phage or to bacterial DNA. . However,. a. preliminary examination of such a phage DNA did not give .. evidence of terminal joining and this may leave open some question as to its ' fine molecular structure. me : : ae Even more specific sequences of DNA are now available by the dissection . .O£ DNA-DNA. and DNA-RNA-hybrids. homologous over a limited range, and by the. ..... reverse transcription of m-RNA's. A technically easier compromise is the Jf partial fractionation of total B. subtilis DNA by differential melting Cf y# ‘Reverse transcription of purified m-RNA from differentiated eukaryotic cells_ may be the most practical general method of concentrating natural genetic specificity to a useful degree. "We “foresee the further possibility of inserting synthetié homopolymer sequences which should sometimes result in the production of the corresponding homopeptides. fuderer a ogersfhave claimed dT )*8uch an effect with TMV RNA by poly-A, but without detailed substantiation - the issue being the persistent replication of such modified sequences; it is entirely reasonable that ehy will be translated into polylysine as predicted by the genetic code). Dr. Elkana's experience in the identification of synthetic homopeptides prompts PHS-398 : Page Rev. 2-69 GPO : 1969 © - 350-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN JO Continuation page her interest in joining this study. Along the same lines, we can, of course, foresee the eventual possibility of inserting artificial genes, e.g. one emulating the code for a transfer RNA as has been synthesized by Khorana's group (and with which Dr. Sgaramella was associated before coming to Stanford). (iii) CO-DONOR AND RECIPIENT DNA. P22. The role of the cohesive ends in the infectivity of P22 may be illuminated by further studies of the transfec- tion system. They presumably are sufficiently important that terminal addi- tions to P22 will be rejected but this must be empirically substantiated. If not, we would be in the fortunate position of being able to extend the genome by a single addition rather than requiring an insertion and a double terminal joining. Dimers, if they were functional, could have the advantage of allowing the disruption of some genetic information by insertions at random positions in one sequence while the other remained intact. With careful handling the cohesive ends might allow non-covalent circle formation, clipping and rejoining of an inserted segment to the two ends of the single molecule. It would be asking a great deal to expect such processes to occur with very high efficiency at an early stage of our investigation. However, it should be possible to contrive genetic selective systems whereby even rare successful insertion, according to this protocol, could be detected. For example, a P22 genome, augmented with bacterial DNA coding for tryptophane synthetase, might form viral clones capable of growing in tryptophane~dependent host-bacteria, (Trans- ducing phage particles have been observed to exhibit substantial "escape synthesis" of the corresponding enzymes even in the absence of specific conditions for induction (48).47/ . For a number of reasons, especially the versatility of genetic functions and analytical methods we would prefer to deal with a bacterial system. The primary bottleneck is the need to discover ways of flush clipping bacterial DNA in a transforming system like B. subtilis. Presumably it will still be necessary to insert new sequences in an ordered series te allow for homologous duplex formation with the recipient bacterial genome on both sides of the insertion. (This is a surmise, though a plausible one, rather than an empirical finding as we have yet to produce terminal additions of sequences to bacterial DNA). This then exhibits substantially the same difficult as was mentioned for P22, It might be surmounted by the use of circularized sequences or through other methods of immobilizing very high molecular weight DNA. For example, we envisage the examination of high molecular weight, folded chromosome complexes (4%)'cf bacteria. If occasional loops of such complexes were clipped, the flush ends might still remain near enough to permit an occasional orderly insertion by terminal joinings of added fragments. Bacterial cells, coagulated with alsohol but broken to expose DNA surfaces would be an easy preparation for large scale trials of such acceptors. Density labelling of the added DNA would facilitate the preferential recovery of pieces of DNA in which insertions had occurred, for these would be expected to have an intermediate density; these concentrated fractions would then be tested for biological activity. Fortunately, these and many other problems that can now be anticipated are amenable to being identified and solved step-by-step. We do not require an improbable global solution to find our path. Episomal DNA» already circularized, can now be transferred with high etficiency in E. coli (4) dnd this may be regarded affording an essentially similar oppor- tunity for the receipt of new sequences that can then be taken up by bacteria PHS -398 Page Rev. 2-69 GPO : 1983 © - 450-360 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page so as to test their genetic specificity. (I will isolatable entity repair the disadvantages that I comparison with B. subtilis during recent years. such investigations. have no hesitation in returning to E. coli if newly found methods for dealing with its DNA as a chemically had perceived it to have by We have retained a very extensive library of strains of E. coli and of Salmonella that could be resuscitated for PHS -398 . Page Rev. 2-69 GPO 71969 O - 250-356 DO NOT TYPE IN THIS SPACE-BINDING MARGIN Continuation page (iv) FUNCTION OF TERMINAL JOINING IN PHAGE The role of a ligase with new tetminal joining activity in T4 infected cells of E. coli is obscure. Ligase-deficient mutants of T4 show increased recombina- tion and a higher incidence of nicks but these phenomena give no hint of a ar, terminal joining function as a natural process in phage replication#/ On the af other hand, one might plausibly speculate that terminal joining plays some role in the integration of double stranded DNA in the course of P22 transduction gga but this would imply that either the genetic information or the enzyme itself is associated. with transducing particles which may be free of viral genome content (“29°5. We intend to study a range of viruses including different Mutants of T4 and of P22 to help ascertain whether any of the indicated func- tions are prominently associated with the terminal joining as distinguished with other capabilites of phage induced ligases. Mutants of P22 capable of differing ey of transduction would be particularly interesting for this purpose ("2 -f-). According to the hypothesis adumbrated previously, terminal joining may be expected to be a feature of some or all of the ligases of eukaryotic cells and our survey will include these sources as well. Existing methrds of assay __ “for the ligase aré tedious and difficult’ and more ‘work for their improvement “| would be a fruitful investhent; furthermore, our survey may well reveal more convenient and richer sources of this enzyme, which is crucial for our further studies. Similarly, existing work bearing on the specificity of the sealing versus terminal joining functions of the ligase will be continued in order to get a clearer understanding of the way in which these two functions relate to one another and of the chemical mechanism of the catalytic process. Much has still to be learned about the relationships of RNA sequences to DNA ligation, an arena expected: to be included in this study. This investigation obviously fits neatly into complementary ones of looking for ways of shaping the ends of DNA molecules of biological interest. (wv) OBSTACLES TO INTEGRATION AND FUNCTION OF INSERTED SEQUENCES 2 “ * The motive for our search for deletions in B. subtilis was to provide,. by ’ studies of transformation using DNA from the intact wild type into deletion mutants, a model of what might be expected to occur when an’ insertion-modified DNA confronts a bacterial genome. As indicated, we have had some difficulty in obtaining deletion mutants, but one or two putative candidates have shown a definite although modest rate of acceptance of wild type DNA, This is con- * “sistent witha Tong ‘tradition of “study of transduction involving ‘deletion mutants |~ in Salmonella. Very recently Adams has reported (2) in some detail on. _ exactly this problem involving a large deletion recognized by arsenate-sensitivity. She concludes that transduction occurs with nearly perfect efficiency but that transformation is impaired about a thousand fold. She concluded "that the _ .. physiological. state of. competence is. at least partially responsible for the - -.-+ exeluston. of: non-homologous DNA sequences. regardless if they are of ‘transférming “|: or transducing origin". The reduced efficiency in transformation may reflect the rather extensive size of the deleted segment; in any case, this finding, although dampening, is not fatally discouraging and more doubtless remains to _ be learned about the conditions that will permit deletion mutants to be efficiently transformed. Precisely the same craftmanship will then be useful in setting up the most appropriate experimental conditions for achieving new insertions. PHS-398 Page Rev. 2-69 . GPO : 1969 © - 350-360 . DO NOT -TYPE'IN THIS SPACE-BINDING MARGIN JX Continuation page Again, we are fortunate to have powerful selective techniques to be able to discover even rare "successful" events out of a large population of failures. Furthermore, a single insertion-transformed clone, when the genetic informa- tion comes from a different species, should be susceptible of unambiguous diagnosis by immunological or eventually amino acid sequence analysis of the protein products (vi) PROBLEMS. Although most of this discussion is oriented toward the perfection of an invaluable technique, this can hardly be achieved without a deeper understanding of the subtleties of DNA specificity and of the cellular mechanisms which are involved in the rejection of invading DNA molecules which may often have pathogenic portent. These investigations, therefore, must lean heavily on what is already known of cellular discrimination against viruses and of the circumstances where this may be expected to break down. By the same token it also bears on the mechanisms of speciation in bacteria and on the most general evolutionary problems. : _ Besides the questions that are inherently incidental to the achievement of molecular translocation and which concern the panoply of enzymes concerned with DNA replication and recombination, there are a number of specific questions ..~ whose. solution. would -be. greatly. facilitated by: the development.of these -tech-— —--- .niques. They would fall generally in two categories: (1) where the ability = to.conduct any DNA transformations with respect to a given genetic trait is already the crucial advance and (2) where the relocation of genetic informa- - tion within the genome is of immediate importance. Under the first heading, we would have a large variety of problems that arise in the genetics of bacterial species for which recombination systems have not yet been described, but. from which DNA can. be extracted and which. DNA is undoubtedly at. the basis of their hereditary specificity. Both headings comprise a considerable range of questions that concern regulatory mechanisms in bacteria. The one of most proximate interest to us, in an extension of a long history of previous pl 7 work, concerns the mechanism of phage’ variation in Salmonella (q% 23 “haf ). BoD. Although it was established long ago that the alternation of flaseliar antigenic specificity is based on an “alternation of state" at a specific locus in Salmonella, we have never been able to ascertain for sure whether this involves a substitution within the DNA sequence or some kind of epinucleic modification, possibly tmiore akin to regulatory phenomena in higher organisms. Any system that would allow a high efficiency of transformation with purified DNA would serve to help solve this problem; for one could determine whether the alterna- tion of state was inherent in purified DNA or not... It is. problematical, how-_ “T""eyer, whether this willbe achieved more readily as a restricted problem in its own right or as an example of gene transfer, say, from Salmonella to Bacillus subtilis. The same remarks apply to the application of these techniques to the .. €tudy of genetic specificity of DNA from eukaryotes by transplanting segments © ‘mation from eukaryotic nuclei could presumably help answer many deep questions about differentiation, diversification in antibody formation, the genetic foundations of differences between normal and ‘neoplastic cells, indeed represent a large scale extension of existing approaches in the genetics of. somatic eukaryotic cells. Some more directly applied implications of these techniques are addressed in section D. PHS -398 Page Rey. 2-69 , GPO : 1969 O - 350-360 ' to-a- bacterium or a virus. Artificial microbial complexes“with genetic infor-— ~ “J DO NOT TYPE IN THIS SPACE-BINDING MARGIN 13 Continuation page ON be ian a Fie fo fire. Beit Ae oth! ns. td NI thoes ol he Ahe fish cabs “beet” ore 4 D 6S GNTRTOANCE SN In t previous paragraphs, I have stressed some of the more theoretical outcomes that might be expected from the technique of molecular translocation. At a more applied level, one should simply say that this should give us a whole new set of tools for the artificial breeding of new kinds of hybrid cells and species, allowing for a general disregard of existing barriers to recombina- tion. It may be a long way before such techniques can be extrapolated to crop ' plants with obvious implications for agriculture; and, longer still to animals; and, there are of course obvious ethical barriers to experimentation in man in this regard. As I have outlined in a recent reflective article (2.5); I believe that it is a great mistake to discuss genetic engineering as if man were the implied primary target: this is no more likely to be true than it has been for the application of Mendelism for genetic engineering. On the other hand, the genetic engineering of bacteria and viruses can lead to the ready availability for therapy and for prophylaxis of biological products which are now vanishirny scarce, to experimentation with new evolutions of amino acid sequences, and to a new repertoire of diagnostic procedures, particularly for situations involving genetic deviation. These "genetic engineering" applica- tions should pose no significant ethical problems since they involve no ..Manipulation of. human.or.prospectively human subjects, but. merely. the extrac- tion of. very short sequences of DNA from a mammal or man and its implantation - in a microbe. The theoretically feasible capacity to produce isolated anti- genic proteins characteristic of pathogenic viruses in innocuous organisms that can be grown cheaply on a lLarge scale would already be sufficient to repay the investment in this kind of work many times over. E. FACILITIES AVAILABLE “ The Department of Genetics and the Kennedy Laboratories for Molecular Medicine have been operating for many years as a well equipped research _ laboratory in this field with all. the principal facilities that are pertinent to molecular biology research. Equipment requirements will have to do mainly » with replacement of specific items’ as they are worn out or occasional updatings of specialized items. In addition, we have been fortunate in being able to assemble some quite sophisticated instrumentation capabilities, initially . with help from the N.A.S.A. and subsequently. from the biotechnology | resources _ “branch of NIH. While these computer and ‘other advanced instrumentation facil- ities are mainly directed to other purposes they have often proven useful in solving specific analytical problems, for example the application of mass ‘spectrometry to the identification of N-chlorocytosine (Sy ). , PHS-398 : Page Rev. 2-69 GPO : 1969 © - 350-360