{Leave Blank) Department of {Leave Blank} Received Date 11-6--58 HEALTH, EDUCATION, AND WELFARE | ¢.1:496 PUBLIC century SERVICE NATIONAL INSTITUTE OF HEALTH at ’ 7 GENETICS (7) Council Assigned Interim Mail Completed Application to: Formerly Division of Research Grants CeZ2157 (C10) Action National Institutes of Health Bethesda 14, Md. APPLICATION FOR RESEARCH GRANT Date__ November 1, 1958. . Application is hereby made for a grant in the amount of gia 4TO for the period from February 1 1959 through“) ~August_31__—«(1959 inclusive (month) (day) (year) . (month) (day) (year) for the purpose of conducting a research project entitled (Limit to 53 typewriter spaces). GENETICS OF BACTERIA Check One: [4 NEW PROJECT (J SUPPLEMENT TO PHS GRANT NO. ee (] RENEWAL OF PHS GRANT NO. ___ «FY REVISION OF PHS APPLICATION NO. ________ Principal Investigator Co-Principal Investigator, if any: Name voshua Lederberg Name 72222 (First) (Middle (Last) : (Firs?) (Middle) (Last) Title " professor of Genetics Title Dept. Genetics Dept. School of Medicine School University or University or Institution Stanford University inchtution Street Address Street Address City and State Stanfo rd, California City and State Name, Title and Address of Financial Officer: Check to Be Drawn as Follows: Duncan I. McFadden, Controller Stanford University Stanford University Stanford, California AGREEMENT It is understood and agreed by the applicant: (1) That funds granted as a result of this request are to be expended for the purposes set forth herein; (2) that the grant may be revoked in whole or part at any time by the Surgeon General of the Public Health Service, provided that a revocation shall not include any amount obligated previous to the effective date of the revocation if such obligations were made solely for the purposes set forth in this epplication: (3) that all reports of original investigations supported by any grant made as @ result of this request shall acknowledge such support; (4) that, if any invention arises or is developed in the course of the work aided by any grant received as a result of this application, the applicant institution will either (a) refer to the Surgeon General for determination, or (b) determine in accordance with its own policies, as formally stipulated in a separate supplementary agreement entered into between the Surgeon General and the grantee institution, whether patent protection on such invention shall be sought and how the rights in the invention, including rights under any patent issued thereon, shall be disposed of and administered, in order to protect the public interest. _ Stanford University Stanford, California NAME OF INSTITUTION ADDRESS CITY AND STATE NAME AND TITLE OF OFFICIAL AUTHORIZED TO SIGN FOR Frederic 0. Glover, Assistant to the President . INSTITUTION (Please Type] . fog: pg wa PERSONAL SIGNATURE (This agreement must carry the actual {use ink} signature of the official whose name appears on the line above). PRIVILEGED COMMUNICATION PAGE |! PHS 398 Form Approved - Loe =e _. Budaet Bureau No. 68-R249,7 Baw OEF (LEAVE BLANK) c-4496 | PROPOSED BUDGET for the period shown on page | NOTE: Under column entitled "OTHER" indicate funds presently PERCENT OF BUDGET . a , ‘ TIME TO BE REQUESTED available or anticipated from other sources, including those from eeNT OM REQUESTEI BTHER own Institution. THIS PROJECT (Omit Cents} Itemize All Positions, Indicating Type, Percent of Time To Be Spent On PERSONNEL: This Project and Names of Pro essional Personnel Selected. . $ : $ J. Lederberg Professor Principal investigato 60 an Stanford Univ. Résearch Associate, oD TOO =ae= . | 6 OOO Research Assistant, M.A. (* T mos. salary; in 100 2800* Preparator B.A. (eludes OAS!, retire=- 100 2100* Di shwasher(s) ( benefits where app! 100 1100* TOTAL PERSONNEL; TF 5 000 PERMANENT EQUIPMENT (See instructions reference itemization of equipment) $ $ Res. microscope, ph. contr. + fluorescence accessories; camera; micromanipulator 2400 Lyophil apparatus for culture preservation 700 incubators and water baths - 1000 TOTAL EQU! PMENT tr «4 100 Capital construction and other equipment of laboratory 85 000 CONSUMABLE SUPPLIES (Itemize] $ $ Chemicals and glassware, Including initial stocks 3 000 TRAVEL (State Purpose) 4 U.S~) $ $ To scientific meeting and other laboratories (prof. per¢) DY 500 OTHER EXPENSE (Itemize) PubTi cation $ w== ([s$ NOTE: The administrative official signing this application may add an SUBTOTAL 13 Loo ES amount for indirect costs. (DIRECT COSTS) |$ , _ , INDIRECT COSTS —— HS PARTICIPATION —=——— IMPORTANT ua” ' 2 O10 Sass Review detailed instructions before BOW POLAR _—SS— e . . ———— computing indirect cost allowance. TOTAL ———— BUDGET 15 k10 aS (OMIT CENTS) $ ——— ESTIMATE OF FUTURE YEARS REQUESTED FROM PUBLIC HEALTH SERVICE INDI PERSONNEL EQUIPMENT SUPPLIES TRAVEL OTHER Ist $ $ $ 2nd 3rd 4th If additional years requested are not contemplated enter "NONE" under total for first additional year. PHS 398 Rev. 9-57 PAGE 2 (LEAVE BLANK) C-4496 i PUBLIC HEALTH SERVICE SUPPORT: Show previous and current Public Health Service grants supporting this project: GRANT NUMBER - ‘TITLE OF PROJECT ‘ AMOUNT PERIOD OF SUPPORT PREVIOUS . 10 yr tetal 2157 to to - Genetics of Bacteria Coo 1946-1958 to University of #83 065 WESCONSIN CURRENT C-2157 CIO Genetics of Bacteria. to University of | The present application 1s for an equi- Wisconsin valent amount (Including cerryever f 13 600 1936-39 C9 to be made available at Stanford ¥.) ALL-OTHER SUPPORT: Excluding Public Health Service, but including that from own institution, list support from other “sources for this project. If none, so indicate. SOURCE TITLE OF PROJECT AMOUNT PERIOD OF SUPPORT cuRRENT ly conml tted3 Pe amt, Capital construction; Dept of Genetics | ~% 150 000 1959 Stanford Univ. sataries and operating costs PENDING applications for additional funds: NSF 5 yr total Genetic recombination In bacteria : 1959-1963 109 500 RESEARCH PLAN’ AND SUPPORTING DATA On the continuation pages provided give details of the proposed plan and other necessary data in accordance with the outline below. Number each page, the first continuation page being page 4. Additional continuation pages, if needed, may be requested from the Division of Research Grants. See detailed instructions before preparing this portion of the application. 1. PHS 398 Rev. 7-56 RESEARCH PLAN A. Specific Aims—Provide a concise statement of the aims of the proposed work. B. Method of Procedure—Give details of your research plan. For each specific aim mentioned in "A" show how your plan is expected to fulfill the aim. C. Significance of this Research—Explain why the results of the proposed work may be important. D. Facilities Available—Describe the general facilities at your disposal. List the major items of permanent equipment, PREVIOUS WORK DONE ON THIS PROJECT Describe briefly any work you have done to date that is particularly pertinent. PERSONAL PUBLICATIONS Cite your most important publications on this or closely related work. List no more than five. RESULTS OBTAINED BY OTHERS Summarize pertinent results to date obtained by others on this problem, citing publications deemed pertinent. Select no more than five. : BIOGRAPHICAL SKETCHES Provide brief sketches for All professional personnel selected who are to be actively engaged in this project. PAGE 3 C-4496 C2157 (clo) RESEARCH PLAN AND SUPPORTING DATA 1AcB. Current work and research plans. On the whele, we plan te continue our studies of mechanisms of genetic transfer In bacteria. Fer the most part, these are direct extensiens of current werk en sexual recombination in Escherichia cell and on transduc- tion by phage In E. col! and Salmonella. Past accemplishments are summarized In the comprehensive summary appended herete. {ft Is net feasible to separate consideration of recent findings, current eperations end future plans.. The fellewing aspects can be expected te have preferential attention by the greup ef students and associates working In the laberatory. (1 may add at this point that Mrs. Lederberg's participation has facilitated the management of a larger program than might etherwise be pessible. ! can therefore plan te spend the larger part of my own time In the laboratory. The students and fellows here are responsible for the main research activity. Nuch ef the technical help we require is te handle reutines ef medium-making and general housekeeping for the cemmen benefit of students and senior investigators. These reutines are quite extensive in the type of work we do. ) a. The nature ef the F cempatibility facter and its relationship to Hfr loci. This study wil! {nvelve the further analysis of a series of Hfe mutants already tseleted. Seme ef the Nfr's have demonstrably different lecations, but de net seem te Invelve rearrangements ef ether bacterial merkers, an impertant peint in various hypotheses ef F/Hfr relatienships new current. Anether appreach is the inheritance ef F in matings, both en masse and in single cell pedigrees. F seems to differ from all other markers [in its centagleusness, as it wlll spread threugheut an Fe culture seeded with a single Fe cell. Hewever, mere detailed studies of this are: . needed. A plausible working hypethes!is, which differs slightly frem that advanced by Jacob and ethers, Is that the F+ mating type carries the F agent as a cyteplasmic factor, while the same agent can become fixed to various chremesemal sites te give Hfr types. Mr. Y. Hireta has discevered that the treatment of F+ cultures with acridine erange results in F- types. Preliminary experiments in micredreplets verify that this is an Induced less and not merely a selection against the F facter. Mere detal led studies are required te determine whether the dye merely inhibits the replication of the F facter er actually destreys it. Studies are also being continued en the conditions ef Its action, fer example, en the identification of a cofacter which is feund In peptene, as clues to its target in the Fe bactertum. b. The wel}=knewn phenomenon ef phase- variation ef flagellar antigens tn Salmonella has been analysed by genetic transduction metheds, with the finding that a phase-determminant is linked te or identical with the H, (phase=2 antigen locus. This determinant escll lates between an acti vé 4 | oe C-4496 and Inactive state. Further studies are directed at 1) the genetic centro! ef this alternation, In menephasic variants, and 2) its pessible centre! by environmental facters. Seme preliminary experiments suggest that tempera- ture shocks cause a slight phase shift, but It has net yet been possible te disentangle it from a pessible differential killing ef the twe phases by heat. A new appreach te the preblem has been furnished by the discovery ef spectal strains ef Salmonella that can be hybridized with E. coli. Or. L. Baron's observations on this polat have been confirmed and strains sult- — able for large scale Investigation of Salmonella by sexual recombination techniques are being developed. These strains should also make it possible, for the first time, to correlate other aspects ef the genetic contre! ef specific functions In Salmonella by transductional and by sexual recomb | r-- ational analysis, Fer example it should become possible te estimate the precise scope of the Individual act ef transduction in relatien to the entire map. c. ONA-mediated transduction (transfermation). The direct transfer of markers by ONA in enteric bacteria would be an invaluable tee! in the advancement of genetic chemistry. in contrast te the pneumececcus and hemophilus, where genetic study for ether reasons Is more difficult, enteric bacteria have se far given negative er indecisive results In the hands ef a nuaber of investigaters, myself Included. My om past trials in this direction have been relatively casual. The technical preblem has become se urgent that a mere concerted effert is new called fer. Since ‘dna’ transferred by phage particles Is genetically effective, the main impediment te dna-transduction may be reasened to be in the penetration of dna particles inte the recipient bacteria. Some ef the variables te be manipulated in this pregram are (1) the test marker, (2) the genetype and the strain of the dener cells (3) the methed ef preparatien and the state of purification ef the DONA, (4) cenditiens of application and pretreatment of the recipient cells, and (5) genotype and strain of the recipient. Existing Infermation en the pneumececcus gives some possible empirical guidepests, but there Is prebably nething better te de than trial and errer, an appreach that weuld hardly be cemmendable for a less urgent technical alm. As to (4) particular emphasis will be laid en the use ef proteplasts and L-colenies as recipients, theugh this ratienale has so far net been substantiated. As te (} and 5) stress will be laid on markers which are transducible by phage with high efficiency, and en systems where recembination by ether mechanisms Is under precise centre]. However, rather than rely tee heavily en a prier! ratienalizations, much weight wil! alse be given te an empirical appreach. One instance ef such en approach was not successful: namely, a screening of some 200 distinct strains ef E. coll as potential recipients. New encouragement for suyeess in this direction comes from the recent observation of Dr. A.B. Kaiser on the transfer of Gal# genes by DNA extrac- ted from lambda. We look forward to a close association with Dr. Kaiser =@ C~4496 whe will be werking in an adjacent laberatery in the Department ef Biecherm istry at Stanferd. d. Phystelegy of mating. The varieus steps ef mating In E. coll may be systematized as fellows: cellisien and agglutination; conjugation, fer- tilfzatien, chromeseme synapsis and cressing-over, segregation. Ouring the past several menths, Or. tL. Cavalli (Pavia, italy) cellaberated with us in an experimental review and theoretical kinetic analysis ef the experiments en interrupted fertilizatien published by Wellman et al., 1956. which are an Indispensable basis fer further studies In this fleld. A cleser leek at each step Is now in erder. For example, thelr analysis ef interrupted fertilization is based en the extrapelatien of time-dependent curves fer the recevery of varieus markers. These curves are semetimes rather shallow and thelr detaltled ferm difficult te analyse en acceunt ef the continued initiatien ef new matings in the cell mixtures. For more precise kinetic analysis the various steps sheuld be more exactly centrelled by environmental facters. We were unable te separate collision frem conjugation; at lewer temperatures petential cenjuga! pairs do net accumulate. One appreach te sefiarating conjugation frem fertilization was a '‘pulseomating' experiment: mating was permitted at very high cel! densities fer one minute; the mixtures were then gently diluted a theusand- feld te allew the pregressien ef cenjugal pairs already fermed, but prehibit new pair fermation. Mewever at high densities, the rate ef mating fol lewed @ square reet rather than the expected secend pewer dependence en teta!l cel) cencentratien. This partly frustrated the design ef the experiment; it may be related te finding that extra female cells added te a mating tended te interrupt matings already in pregress, suggesting seme ferm ef active com- petition fer the active sites en male cells. A mere promising Jead was the finding that perledate in certain cencentrations weuld temperarily de- masculinize male cells, neither killing them ner interfering with the pregressien of matings already started. This strengly suggests that a periodate-sensitive carbehydrate is invelved In the specificity ef the initial mating reaction, and chemical cemparisens of male and female cells are prejected sleng with trials ef varieus pelysaccharases te try te test this suppesitien. e@, Cytegenetics ef fertilizatien. (Mr. A. T. Ganesan). Apart frem the inittel demenstratien ef cenjugal pairs classical metheds have net been given their full due in the study ef fertilization; mainly fer went ef assistance by suitably trained advanced student er fellew. Mr. Ganesan's backgreund In yeast cytelegy and genetics (mainly at the Carlsberg Laboratery at Copenhagen) is mest premising In this respect. The eriginal phetegraphs gave seme hint ef the passage of Giemsa-pesitive material but a critical analysis still has te be made. Clesely cenneacted with this will be efferts te essay the transfer ef P22 labelled ONA from !abel led male pretoplasts mated to female reds by means cf the micro-radiegraphic ‘star! | methed of Levinthal. We have verified that male preteplasts retain their mating com- petence and that progressive fertilization can be interrupted without dis- turbing the female member by lysing the male conjugant in distilled water. The very few unlysed (dead?) males sheuld be recognized by very high star counts; fertilized female cells which ean be washed following enzymatic “> 6 C- 4496 extractions, if needed, should have a star count reflecting the Input of labelled DNA. This experiment should permit a final verification of the Jaceb-Wel lan hypethes!s ef pregressive fertilization, and the cerrelatien of quantity of DNA with genetic length. Oorrferaeen: facilities at Wisconsin were: anpromising fer this leng-planned experiment; it may be done. | ot at Stanferd «: In collaberation with Dr. $. Lederberg of Brown University. Geren and Skasr have published experiments on P> transfer in mass matings. f; Recambinational analysis of galactese mutations. (£. M. Leder- berg). The complex of clesely linked Gal mutants affecting the fermenta- tien of galactese eccuples a promising place in biochemical genetic cerrelatien fer several reasons: (1) the identification of sequential defects in specific enzymes by Kalckar; (2) the scope and simplicity of analysis of these fecters by ‘high frequency’ transduction by the phage lambda; (5) the avallability ef more than ene hundred nonrecurrent mutants. Many of the mutants fit Inte a simple picture, whereby a set ef mutants failing into one cistren (pesitien effect greup) cerresponds te ene of the three enzymes (kinase, transferase, epimerase) In Kalekar's scheme. Hewever, a number of anomalies have appeared, @.g., the mutant Galx which behaves recembina- tionally as a peint mutant, but impairs the fermation ef all three enzymes, and overlaps at least twe ef the cistrans; anether mutant Galop belengs ce nelther of the other cistrens (i.e. ferms galactose-pesitive transhetere- genotes with each ef them). The validity of the concepts of simple cistren- enzyme relationships (i.e. of linear ceding) so facilely accepted by many workers today, needs to be tested vigorously and extensively. Some indi- cation that Gal, is dtructtraliyrabersant has'been found from expetimentsts Tra whieh variou§ Gal muiants are mapped by ‘'timing' in interrupted fertili- zation. Galz ts delayed several minutes whereas most of the Gal mutants fall within One minute ef one another. The time-mapping, which requires considerable technical imprevement te facilitate its use far short Inter= vais, is alse being eppiied te determine whether each cistron maps compactly witheut everlapping the lec! ef ether cistrens. Other efferts to map the sequence of Gai mutents have occupied a great deal ef eur time during the past two years, but have been frustrated by a high coincidence of crassing~ ever in three and feur pelat tests. tnfertunately, few known markers are clasely linked te Gal; extensive surveys te find ether auxetrephic markers that would accompany Gal in transduction by lambds have failed. Parallel studies are under way with complexes ef Lec (lactese) and Ara (i-arabinese) mutations. g- Prephage relationships in lembda transduction. The findings by Campbe!l, Arber and ethers that auxiliary phage greatly increases the efficiency of transduction remeves the main support fer our previeus cencluston that the same phage particle may carry the Gal markers and an intact phage. Studies on mere complex systems (syngenetic recipients; 27 transductions to bacteria lysogenic for related phages) stil] leave open the possibility of at least an occasional association either in the original transduction, or in the reorganization of the [Input material in the hetere= genote. These studies will be resumed in connection with the mapping of the excgenotic markers in hetersgenete crosses, as mentioned abeve. 7 be 10. ie 3o Ur 4490 Significance ef this research. The principal motivation ef this research pregras |s further under- standing of cellular heredity. Findings in bacterial genetics may alse be expected te have an important bearing en connected areas of taxeneny, ’ phystolegy, ecology, and se forth. Altheugh we are net immediately concerned with practical applications in medicine, further Improvements in medical practice must depend on our fundamental knewledge ef etielo- gical agents of disease. The analysis of genetics ef microergan! sms alse plays @ rele in the comprehension of the genetics ef viruses and of higher erganisms and thelr cempenent cells. Facilities available. After eo shert Interval of tenporary eccupancy ef quarters ina the Biephysies Bullding at Stanferd University, this program will be heused in a greup ef laberatery reoms in the new medical center. We will be working In clese asseciation with the Separtment ef Blocheal stry and will share with them the use of, and responsibility for maintenance of many general facilities as well as highly special ized equipment. The basic items of equipment are being assenbled frem suppert from University and other seurces as well as the present grant and we sheuld be well equipped to recommence our research pregram seen after moving Inte the temperary laberatery February !, 1959. Previeus work. See sumeary repert appended. Joshua Lederberg. 1947. Gene recombination and linked segregatiens ia Escherichia cell. Genetics 32:505-525. deshua Lederberg, Esther M. Lederberg, N. BD. Zinder and E. R. Lively. Recombination analysis ef bacterial heredity. 1951. Cold Spring Harber Symp. 16:413-443. Jeshua Lederberg, L. L. Cavalli, and Esther M. Lederberg. 1952. Sex compatibility in Escherichia cell. Genetics 37:720-T. Esther M. Lederberg and Jeshua Lederberg. 1953. Genetic studies ef lysegenicity In Escherichia coll. Genetics 36:51-64. deshua Lederberg. 1956. Linear Inheritance in transductional clones. Genetics 41:845-871. Results ebtained by ethers. Recembinstien genetics ef bacteria is new tee active a field to be summarized briefly. Varieus aspects are dealt with In recent sympesia, ¢.g., Celd Spring Harber Sympesiua fer 1956, and the Wong. uw Pratt institute Sympesium on the Chemistry ef Neredity, Baltimore, 956. 5. 6. C-4496 Blegraphical sketch. Principal investigater - Jeshua Lederberg, b. Montclair, N.Jo, Fay 25, 192°; B.A. (Zeelecy) Columbia Gellege, 194%. Colusbla University, Co’ lege of Physiclens and Su s (medical student) 1944-1986. Yale University, (Hicreblelegy), 1986-194 > Ph.D. Uiversity ef Wisconsin, Department ef — Genetics: Assistant Prefesser, 1947-1950; Asseciate Prefesser, 1950-195). rrereeees ef Genetics, 1954-1958; Prefesser and Chairman, Medical Genet: 5, 1957-19) ° STANFORD UNIVERSITY Medical Schee!: Prefesser and Executive Head, Depart- ment ef Genetics, 195Qeoce-. University of Callfernia, Berkeley. Visiting Prefesser ef Bacteriolog: , Summer, 1950. Melbourne University (Australla). Fulbright Prefesser ef Bacterielegy, Spring 1957. dustification ef specific budgetary requests. This application Is fer the resumption at Stanford University of the pregrem that has been undertaken at the University ef Wiscensin with public Health Service support under grant C-2157 (CIO). The budget centemp! ates @ program of the same scepe as has been supported by the NIM since 1952 taking acceunt of Increases necessary en acceunt of salary and cost advances. Fer several years we have had cellateral suppert frem the Natlenal Sclence Foundatien and a request is pending te them fer renewal at Stanford tniversity. The request te NSF was enlarged In erder te take account In part ef the expiration ef suppert frem the Reckefeller Feunda- tien and the Wiscensin Alumni Research Feundation. It may be necessary at a later date te file a supplemental application te the NIH In erder te continue the pregran at the level at which it has been carried eut at Wiscensin but this is being deferred until! we can better assess our needs at Stanford and the present application cevers enly those funds released by the termination ef the preject at the University of Wiscensin. The principal Investigater expects that the present project wil! centinue te eccupy the larger part ef his time. nether request te the NiH Is pending primarily fer the suppert of Or. Gustav: Nessal in a pregram fer the study ef antibedy fermatien In single cells. Alse pending is a request fer a substential graduate research training grant. tf this Is successful the suppert ef graduate students as research assistants en the research pregram will be met frem that grant with the exception of a small number ef ferelgn nationals. in anticipation of this transfer, many expenditures en C-2157 (C10) have been deferred and the persenne! of the laberatery at Wisconsin has been kept te a minimum during the present year. in consequence the entire allocation frem that grant fer the current budget year will be available tegether with a small carryover frem previeus years. This, happily, may allow of the necessary initial capital expenditures needed to get the laberatory underway. The estimate for future years cerrespends te the ? & C-4496 comaltments already extended by the NIM fer C-2157 (CII-CI4). These esti- mates for future years contemplate a pregram ef the sane scepe as that new being supported with a small allewance for increased cests by the third and fourth years. 10