BRL eee DEPART My OF HEALTH, COUCATION, AND WELFA PUPLIC HEALTH SERVICE RE CRANT APPLICATION SECTION 1 D. A. Glaser Form Approved Budget Bureau No. CER LEAVE BLANK NUMBER TYPE |PROGIAM REVIEW GHOUP FORMERLY COUNCIL (Avoata, Weer} DATE RECEIVE TO CE COMFLETED OY MRIRCIPAL INVESTIGATOR fitems T through 7 and THA} yt. TITLE OF PROPOSAL (Do ast exceed $3 typawriver spaces) Scanner-Coapuver Irnvestigetions of Biological Svatems- 2. PRINCIF.AL INVESTIGATOR 3, DATES OF ENTIRE PROPOSED PROJECT PERIOD (This spplicsi OA, NAME (Last, First, Initiel) : FNOM THROUGH Glaser, Ponsld A. O11 June 1075 Fl May TORD DB. TITLE OF POSTION. 4, TOTAL DIRECT COSTS HE. [BT DIRECT COSTS REQUESTEL Professor of Physics and Molecular Biology QUESTED FOR PERIOD IN ITEM 3 6,124,938 | _i,f ’ FOR FIRST 12-MONTR PERI 1, 646,655 2C, MAILING AGORESS SHBG cay, Site, Zip Code} Denartment of Molecular Biology University of California Berkeley, California 94720 6. PERFORMANCE STTETEY (See instructions} University of California Berkeley, California 94720 — 20, DEGREE 2E, SOCIAL SECURITY NO. 2E.TELL Paces Coda TELEPHONE f o ENSION PHONE DATA LATS 6)2 14410 “a. BEAR IMEAY, SERVICE, LAEORATORY OR EQUIVALENT {See f; nstrustions) Virus Laboratory . + ZH, MAJOR SUEDIVISION (See frstructions} * Collerce cf? Letters and Science 7 Hesearen trveiving Wunisn Subjects (900 instructions) ABS HO §{C] YES Approved: C.(E] Ves - Pencing Review ete TO BE CONVLETED OY RESPONSIBLE ADMINISTRATIVE AUTHORITY (items & throuah 13 an 8 Inventions (keaava! Applicants Only + See fastryctions) ACIJNO 8.[(C) VES — Not previously reported C. CAIYVES — Previousty raported nt ThE) 9, APPLICANT GRGANIZATIGONIG) (Sce fasiructions) Vi. TYPE OF ORGANIZATION (Cncek anpicedls item!) The Regents of the University of California (FEDERAL CO] stare (CO tocalL COOTHEN (Specify) e/c Campus Research Office MLL Vireeler . 12, NAME, TILE, ADDRESS, AND VELEPHONE NUREER OF University of California OFFICIAL tN BUSINES 5 OFFICE WHO SHOULD ALSC gE NOTIFIED If AN AWARD IS MADE . Berkeley, California $4720 . . Mr. August G. Manga, Manager Carpus Research Office - MIL Wheeler, University of California TO RAGE, TITLE, ARD TELEMIONE NUIMSER OF OF FICIALE) Berkeley, California SIGWING FOR APPLICANT ORGANIZATIONS) Telephon umrct. PSIBERTTEY ORG i WICAVIGRAL 5 FOR INSTITUTIONAL GRANT PUSE Cees: ve fastructians} Bionedical Seiences Support Grant. . Telephone Sarcber (a: 14, ENTETY HUNBER (Formerly PHS Account Number) hei7o Poa 16, CENT FICAT iON AND ACCUPTANCE, knowledea ond ercont, 93 te any arent awarded, the obligation te comply ve We, the uncselgaad, certify that the ¢ tatements herein eru truo and comple to the best of our ith Public Health Service turms and conditions In effect ot the tive of ihe marr’, GIRS TOS BoA Snr ae nc (Si pnetuces be quad en . oN. orginal copy only. GB. SIGNAT Ricwlab bor SR NAMED IN TTEM 40 DATE - Lawl? ony Uen bok,” tpsepotpreat coma nor ho etn semngetite RedState shee vanes ee ee eee REMOVE AND USE FOR DRAFT COPY “Donald A. Glaser, ‘John Bercovitz, D. A. Glaser SECTION 1 DEPARTMENT OF HEALTH, EDUCATION, ANDO WELFARE LEAVE BLANK ' PUBLIG HEALTH SERVICE PROJECT NUMBER RESEARCH OBJECTIVES , NAME AND ADORESS OF APPLICANT ORGANIZATION University of California Bertteley, California 94720 NAME, SOCIAL SECURITY NUMBER, OFFICIAL TITLE, AND DEPARTMENT OF ALL PROFESSIONAL PERSONNEL ENGAGED ON PROJECT, BEGINNING WITH PRINCIPAL INVESTIGATOR » Professor of Physics and Molecular Biology, Virus Lab. Assoc. Development Engrg., Virus Laboratory , Acst. Development Engrg., Virus Laboratory Assoc. Development Mngrg., Virus Laboratory » Principal Programmer, Virus Laboratory Post-Grad. Research Biophysicist, Virus Laboratory Asst. Develop. Energ., Virus Laboratory » Sr. Development ingrg., Virus Laboratory » Principal Development Ingrg., Virus Laboratory (cont. on p. Ronald Baker, dames Berk, : Fraser Bonnel John Couch, Ted Fujita, Robert Henry, Leif Hansen, TITLE OF PROJECT Scanner-~Computer Investigations of Biological Systems USE THIS SPACE TO ABSTRACT YOUR PROPOSED RESEARCH, OUTLINE OBJECTIVES AND METHODS, UNDERSCORE THE KEY WORDE (NOT TO EXCEED 10) IN YOUR ABSTRACT, - Large scale genetic and physiological studies of bacteria, yeasts, and animal cells grown in tissue culture will be carried out using recently constructed automated eguapment and compyter-directed patvern recognition techniques. By automatic exami- nation of up to 10” colonies in'a batch, rare mutants will be isolated and partially characterized, mutagenic effects of chemical and physical agents will be measured , ever, at low dosés, and genctic recombination frequencies measured accurately for mapping purposes. Mutants for detailed studies of DNA synthesis in BE. coli and B. subtilic will be isolated and partially characterized. Mutants of E. coli, S. typhimuriun and Saccharomyces cerevisiac will be isolated vor study of biosynthetic and degra- dative pathways and for analysis of the mechanisms of genetic recombination. Genetic maps of E. coli, Saccharomyces cerevisiae, and some manmalian cells will be enlarged. Feasibility studies of automatic recognition of bacterial and fungal pathogens in medical and public health applications will be extended. Mutagenic effects of food additives and other enviromental chemicals will be tested in several bacterial, yeast, and animal cell systems. Carcinogenic effects of chemical and physical agents including ionizing rediation will be measured using animal cells. Interactions of hormoncs and other agents with tumor cells grown in tissue culture will be examined SO investigate the biochemical mechanism of the interactions and to test possible anti-neoplastic effects of a variesy of substances. Mutant tumor cells sensitive to some agents and resistant to others will be isolated for further study. Screening. programs may be undertaken when feasible for iwutagens, carcinogens, anti-necplastic agents, and effects oF low doses of mutagens and. ionizing radiation: Additional instrumentation will be constructed as needed. Pe bat wxe fil . : Donald A. Glaser Condsvatioa pou 2g ow ‘Privileged Communication... Professional Personnel (continued) Larry Johnson, é _— Assoc. Devel. Engrg., Virus Laboratory Alex Para, sst. Devel. Engrg., Virus Laboratory ve Ale Ur Laser ~ FROM THROUGH MONTH P a DETAILED BUDGET FOR FIRST 12-MONTH PERIOD June 1, 1975 May 31, 1980 ip yh ae DESCRIPTION (Iremizo) rime on AMOUNT REQUESTED (Omit cents} PERSONNEL EFFORT FRINGE AS, Y NAME TITLE OF POSITION we en BENEFITS TOTAL cca. [PRINCIPAL INVESTIGATOR (See details on sitttached p, 34) 505.872 CONSULTANT COSTS Occasional engineering consultents for special technology 1,500 EQUIPMENT (see details on atteched p. 4b) 828, 200 SUPPLIES ~ “(See details on attached p. 3c) 204 , 642 ._,. One major trip to Hast Coast for professional persons to DOMESTIC . ho attend a major corfermce (Gordon,CSH (52,000); attendance TRAVEL me SCORED by professionals and grads at local conference (Arrowhead, ~ Biophysics); confer with colleagues and equip.suppliers. 4,009 PATIENT COSTS (See instructions} . ($1 , 000 } ALTERATIONS AND RENOVATIONS THER EXPENSES (ftomize) (See details on attached pn. 3c) 103,451 TOTAL DIRECT COST {Enx. Page 1, Item 5) (Enter on Page 7 a 1,616,655 inbIRECT” saws DATE OF DHEW AGREEMENT: Cwaiveo } cost sha 5 F273 ‘ (C] UNDER NEGOTIATION WITH: (Sea Instructions) st ____% TOC = : FO THIS IS A SPECIAL RATE fee. off-site}, SO INDICATE, NIH 398 (FORMERLY PHS 398) . PAGE 3 Rev. 1/73 ThE ee yea ‘See §Steacgemdpea a bE. ee t | PERSONNEL “Privileged Communicatio — Za -. _ Donald A. Glaser... , , ~ Fringe 'Name Title of Position @Time Salary Benefits Total a Donald’ A. Glaser.. Principal Inves. 2@mos. 8,652 1,296 9,948 iMechanical Engineers — | Leif Hansen | Prin. Dev. Engrg 100 25,224 3,026 28,250 John Bercovitz Asst. Dev.Engrg-II 100 «14,748 1,770 . 16,518. { larry Johnson | ‘Assoc.Dev.Engrg.III 100 18,792 2,255 ~ 21,047 | Larry” Henderson Draftsman I 100 8,628 - 1,035 9,663... ;Shop and. Maintenance J _ * ; Walter Debold Prin. Lab.Mech.V 100 16,236 1,948 18,184 Lloyd Davis Prin.Lab.Mech.y. 100 16,236 1,948 18,184 James Munger lab.Asst.II » 100 8,196 983 9,179 | X Lab.Asst.I 100 7,560 907 8,467 xX (2) Lab.Helpers 100 «612,624 1,514 14,138 ‘Computer Programming - : ! Fraser Bonnell ‘Prin.Progr.V 100 «24,612 = 2,953 «27, 565 | x \ ‘Sen.Progr. 100 16,656 1,999 18,655 ; x . Programmer 100) 12, 444 1,493 13,937 !Computer Operations . - | Robert Henry Sr. Dev. Engrg.V 100 = 2k, 012 2,881 26,893 Xx(2) Computer Operator 100 19,906 2, 388 22,294 | tnetrumentation ; ; Ronald Baker Assoc.Dev.Rnerg.V 100 = 20, 748. 2,489 23,237 {| James Berk Assoe.Dev.Engre.I 100 17,052 2,046 19,098 | Ted Fujita Asst. Dev.Engrg.V -100 17,052 2,046 19,098 | Alex Pare . Asst. Dev. Engrg.I 100 14,040 1,685 15,725 “or. Pat Donahoo’ ‘Tab.Asst.I °° “100 -.7,560- 907 8,467 Biological Operations . . ~ ‘ i = John Couch Asst.Res.Biophysicist 100 15,290 2,294 ~17, 584 | Philip Spielman -Staff.Res.Assoc.II 100 = 14, O40 1,685 15,725 i Marilynn Brook. -Staff.Res.Assoc-II 100 ..13,0h4. 1,565... 14, 609 | Carol Greiner Staff.Res.Assoc.I 100 «12,144 1,457 13,601 i Eva Bennett Lab. Asst. II 100 8,208 985. 9,193 James Colby _ Lab.Asst.I 100 7,272 872: 8,144 Xxx (3) Post ~Doctorals 100 35,568 5,335 40,903 XXXX (4) Grad.Students 50% - 9 mo 23,292 2,796 26, 088 100% - 3 mo , ‘Administration and Procurement . o . '- Madeline Moore "Adm. Asst. II 100- 10,248 ~~ 1,230 (11,478 , ’ Total Salaries 450,084 55,788 505,872 Hi a ON Oey tee erm Rn “He GS Last { 8 ts" Sy lee ath Privileged Communteation 4 meet 9 a settee tame n= Caring sion oss « BD = EQUIPMENT PDP 10-I System -KI-10 _MF-10 RP-10 RP-03 DF-~10 “TM-108 TY-LO TD)~10 TU-56 DC-LOA. DC-10B LF-0F _ VB-10C PDP-11 Processor Memory (2)64K words Disk Control Disk Drives(3) Data Control for — Disk and MagTape(2) Mag TapeDrive Control Mag.Tape Drive (2) DEC Tape Control DEC Tape Drives(2) DataLine Scanner Data Line Group, 8 lines Line printer, 1250 lpm ~ Graphic display Controller for flying-spot scanner, (18 bits) “Unit Cost 240,000 - 80,000 ~ 26, 000 20,000 + ete 14,000 20,000 25,000 15, 300” 4.700 10,000 5,500 47,500 35, 000 30,000 ~ Donald A..Glaser . 4 Total 240,000 160, 000 26,000 60,000 -28, 000 20,000 50,000 15, 300 9,400 10,000 5,500 - 47,500 35,000 30, 000 Sub-total (PDP 10-1) 736, 700, PDP 10-I (Sortvare package/p. a.) Laser h watt tunable blue laser — (Spectrophysics Model 164) Flying-Spot Scanner--speed up PDP-11 ~ controller : New faster yoke and A/D converters PDP 10 to PDP 11 direct memory bus Total Equipuent "5,000 49,500 30, 000 10, 000 _ 37,000 828, 200 Donald: Aww -Glaser rm omens Var as wd se g2> FREE 7 tie oe we we od hPa tim arg ce : ag + ate Se Me TOTAL OTHER - Consineeten cege ea 3e eo - veneer _Priyileged Communication. = veneer SUPPLIES , : Software notebook updates (3300); softuare updates subscription (31,100/yr); teletype paper, printer . “paper, Calcomp plotter paper ($1,300); Mag tapes ($300); “DEC tapes (#00). . ~ 3,400 “Petri dishes at $25/case (500 dishes per case), oe 2 cases per week x 50 wks = 100 cases x $25. "2,500 Agar at $1. 30/liter; 0.030 liters per dish; 1000 dishes per week x 0.03 liters/dish x 50 weeks x i $1.30 1,950 ‘Agar for Cyclops trays~+20 trays /e x 1.5 Liters/ oo tray x 50 weeks x $1.30 | 1,950 Agar for Dunbwaiter--2 e xperiments/vk x 256 trays xo Lo 1.5 liters x 50 weeks x $l. 30 liter 49,920 . Miscellaneous arugs, chemicals, nutrients and glassware 7 5,000.” Film and Development $0.18/ft for 35 mm filn ; Cyclops--20 trays/wk x 50 wks x 32 sauares/tray x 6 photos/square x $0.18 ¢ 8 photos/ft. 4,320 Dumbwaiter--256 trays/expt. x 100 ‘oxpts/yr x 32 squares /tray x 6 photos/square, x $0.18 = 8 photo2/ft . ; Po . 110,592 , Miscellaneous small electronic, mechanical and , optical parts for constructing laser selector-inoculator electronic controls, - and new cell manipulation devices 25,000 TOTAL SUPPLIES $204, 632 OTHER EXPENSES a, _ Computer maintenance ($2, gl /no.--see De 3a) - 35,376 - EE Machine Shop (2,000 hrs. at $11/hr) - 22,000 LBL Machine Shop 20,000 Machine shops (special jobs on and off campus ) _ 10,250 LBL Supplies 9,600 Phones . 2,700 Xerox 1,800 . Page charges, 15 pp at-$75/page | 1,125 Publications - professional journals 500 Mail 100° $103,451 Donald A. Glaser Moscouttion vane = Dd = Privileged Comaunication Te ements eter oes ae net mn em emer a ae a ee ee ow wane Sree eee pr neeee ‘Corapuser Maintenance (continued from ps3) Maintensnce per mo KI-10 Processor ’ 550.00 MF-10 888 .00 RP-10 79.00 RP-03 510.00 ° ‘DF-10 134.00 TM-10B 43.00 TU-l.0 316.00° TD-10 _ 20.00 TU-56 . 68.00 DC-i0A 19.00 DC-~1OB 18.00 LP-10F 153.00 VB-LOC 150.00 20 tha NS nT ” ad “Total per mo 2,948.00 a we pe a me v f ret Fe Fy tee ys 4 at oom. GI a * - rid + m. aren a ~ + whe wot ™, fee ee i. a fo fs aoe o 4 @ tes fa CK : SESS vs Pg Te LS 20 ge in? a Y its ge> BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUSLIC HEALTH SERVICE DIRECY COSTS ONLY (Omit Cents) IST PERIOD | ADDITIONAL YEARS SUPPORT REQUESTE Dif Phis application only) DESCRIPTION GAME AS DS. . . TAILED BUCGETIT 2ND YEAR 3ROD YEAR 4TH YEAR 58TH YEAR GTH YEAR 7TH YEAR : “eee . * . ¥* . Penson 505,872 | 556,459 |612,104 | 673,214 {7h0,645 CONSULTANT COSTS 1, 500, 1,650 1,815 |. 1,997 é,197 Unctade fees, travel, etc.) ‘| eourpvent “ 828, 200 75,000 | 75,000 5,000 | 75,000 “| supPLIEs | 204,652 «| 225,095 27,604 | 272,364 299, 600 oe DOMESTIC 4,000 5,300 | 3,630 3,993 4.392 FOREIGN , : PATIENT COSTS LTERATICNS AND RENOVATIONS OTHEN EXPENSES 103,451 | 123,796 | 125,175 (237,692 [151,461 ’ TOTAL DINECT COSTS P6465 655 975,300 [1,065,328 |1, 16%, 36041, 273, 295 TOTAL FCR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1, Item 4) ————» | $ 6,124,938 — REMARKS: Justify aff costs for tha first yeer far which the reed may not be obviews. For future years, justify equipment costs, as well as any significant increases in any other category. If a recurring annual increase in personnel costs is requested, give percentage. (Use continustion page if necded.) . 4 Cost of living . figured at 10% By June 1975. - Hach consecutive year figured at 10% increase for cost-of-living increase and inflation . a oo . Employee benefits figured at 12% for non-academic; 15% for academic salaries. SECTION Il — PRIVILEGED COMMUNICATION -?- = , BIOGRAPHICAL SKETCH (Give tha following information for olf professional personnel fisted on pags 3, Seginning with the Principai investigator. Use continuation paces end follow the srme generel format for bach person. NAME TITLE BIRTHDATE (Ma., Day, Yr.) 7 ‘Professor of Physics and’ . =~ Donald ‘A. Glaser ; Molecular Biology 9/21/26 PLACE OF BIRTH (City, State, Country} PRESENT NATIONALITY f/f non-U,S citizen, SEX indicate kind of visa and expiration date) Cleveland, Ohio, USA USA (3 Mate (] Female EDUCATION (Begin wath bsecalaureste training and include postdoctoral) _ INSTITUTION AND LOCATION DEGREE CONFERRED sclentir iC Case Institute ofTechnolorgy . BS 1946 Mathematics ana Physi California Institute of Technology PhD 19h9 woo. " Case Institute of Technology Sc.D. 1959 " " HONORS Henry Russel Award, 1955; Charles Vernon Boys Prize, 1959; American Physical Society Prize, 1959; D. Sc., Case Institute of Technolosy, 1959; Nobel Prize (Physics), ‘1960; Ellio Cresson Model (Franklin Institute) 1961; Alumni Distinguished Service Avard (Cal.Tech.) 1967; Gold Medal Avard (Case Institute of Technolory) 1967. MAJOR RESEARCH INTEREST ROLE IN PROPOSED PROJECT Cell genetics and control mechanisms Principal Investigator RESEARCH SUPPORT (See instructions} NIH Grant GM 19439 bf/1/73 = 5/31/74 Genetic Control of Cell $e, 262 ‘ Physiology and Structure NIH Grant GM 1324 09) «= 6/1/73 - 5/31/74 Scanner-computer investi- $405,698 mo . gations of biological . systems RESEARCH AND/OR PROFESSIONAL EXPERIENCE (Starting with present position, dist treining and experience relevant to area of project. List ali or most representative publications, Do not exceed 3 pages for each individual.) Visiting Professor of Biology MIT 1961-62 Miller Professorship UC Berkeley 1962-64 Consultant Brookhaven National Lanoratories, Argonne National Laboratory, and a varicty of other laboratories and agencies on scientific and instrumentation ~ problems in physics and biology. i. C. B. Ward, M. W. Hane, and D. A. Glaser, "Synchronous re-~initiation of chromo- rome replication in BE, coli B/r after nalidixic acid treatment," PNAS 66, 365 1970). 2. C. B. Ward and D. A. Glaser, “Control of initiation of DNA synthesis in BE. coli | B/r," PNAS 67, 255 (1970). ” 3. C. B. Ward and D. A. Glaser, "Correlation between rate of cell growth and rate of DNA synthesis in Escherichia coli B/r," PNAS 68, 1061 (1971). 4. oD. A. Glaser and C. BY Ward, “Computer identification of bacteria by colony morphology", Frontiers of Pattern Recognition, Acad. Press, N. ¥. (1972). D- Jd. Couch, J. Berk, D. A. Glaser, J. Raymond, and T. Wehr, "Automated recognition of bacterial strains by analysis of colony morphology", Proceedings of the 13th International Congress of Genetics, Berkeley, California, August 1973. 6. J. Raymond, J. Couch, D. A. Glaser, and C. T. Wehr, “Automatic selection of conditionally defective mutants of micrcorganiems," Proceedines of the 13th (continued) WIH 99¢ (FORVERLY PHS 392) Rey. 1/73 : ak veers ew al PR 2 NT “NM wy 7? afte ial OU ae me tae ee . OE WAeek ay a theese _Cortiewatios mye 1 6. Donald A.. Glaser Privileged Communication =» 7. C. T. Wehr, L. Waskell and D. A. Glaser, “Isolation and characterization of | ' cold-sensitive DNA mutants of Escherichia coli K12", Proceedings of the 13th International’ Congress: of Genetice, “Berkeley, California, August 1973 « 8. RM. Burger and D. A. Glaser, "Effect of nalidixic acid on DNA replication by toluene-treated Escherichia coli", Proc. Nat. Acad. Sci. 70, 1995 (1973). 9. D.-L. Parker and D. A. Glaser, "Chromosomal sites of DitA-membrane attachment . in Escherichia coli", submitted to J. Mol. Biol. September 1973. 10. D. L. Parker and D. A. Glaser, "Effect of growth conditions in DNA-membrane | attachment in Escherichia coli; in preparation. il. A. H. Dougan and - D. A. Glaser, "Rates of chain elongation of ribosom fal RUA molecules in Escherichia coli", submitted to J. Mol. Biol., 1973. 12. L. Waskell and D. A. Glaser, “The isolation and partial characterization of mutants of E. coli with cold-sensitive synthesis of DNA", in preparation. 13. D. A. Glaser, "Some effects of ionizing radiation on the format ion of bubbles in liquids", Phys. Rev. 87, 665 (1952). oS Us. D. A. Glaser, "Bubble chamber tracks of penctrating cosmic ray particles", Phys. Rev. 91, 762 (1953). ; of 15. D. A. ‘Glaser, "Progress report on the develépnent ‘of bubble chambers" 'y Nuovo Cimento 2, suppl. 2, 362 (1954). 16. D. A. Glaser and D. C. Rat, "Characteristics (of bubble chenbers", Phys. Rev. 2b hl (1955). | 17. OD. A. Glaser, “the Bubble Chamber", Scientific American 1955. . 18. J. L. Brown, D. A. Glaser, and M. L. Perl, "Liquid xenon bubble chamber", Phys. , Rev. 102, 586 (1957). | 19. OD. A. Glaser, D. C. Rahm, and ¢. Dodd, "Bubble counting for the determination . 4 ’ . of the velocitics of charged particles in bubble chambers", Phys. Rev. 102, 6, -1653 (1956). oe , So 20. OD. A. Glaser, Decays of strange particles, Kiev. Conference, 1950. 2l. D. A. Glaser, et al., "The neutral branching ratiog of K° particles", Phys. Rev. Tetters en . - 22. D. A. Glaser and L. on Roellig, "Elastic +p and p-p scattering at 1.23 Bev/e." . Phys. Rev. 116; 1001 (1959). - we 23. D. A. Glaser et al, "Direct proof of, 3} neutral decay", Phys. Rev. Letters 3 51 (1959). — 4 a , 2h. oD. A. Glaser and . I. Yettenbe Te, "ay automated syste for the growth end analysis of “arge mimbers of bacterial colonies using an environvental caamter Ca at poe 26. eit ae nee nite waienenin © 8 te esaey » 25. 2T. ; 28. 29. 30. 31. Cartleuation aga To + Donald.A..Glaser__...._... Privileged Communication ‘ —- a - . en eee and a computer-controlled Tying-spot scanner", Ann. N.Y. Acad. Sci. 139, 2h5 (1966). D. A. Glaser, "Biologic al objectives and stratery for the design of | a space vehicle to be landed on Mars." Chap. 18, Biolozy and the Exploration of Mars, Nat. Acad. Sei. Nat. Res. Counest publication, 1900. D. A. Glaser, Je MeCarthy and M. Minsky, "The automated “piological laboratory, B. Wolf, A. Newman, and D. A. Glaser, "On the origin and direction of. peplica« tion of the E. coli K12 chromosome", J. Mol. Biol. 32, 611 (1968). 2 M. L. Pato and D. A. Glaser, "The origin and direction of replication of the chromosome of Escherichia coli B/r. Proc. Nat. Acad. Sci.. 50, 1268 (1968). C. B. vard and D. A. Glaser, "The origin and direction of DNA synthesis in E. coli B/r.- Proc. Nat. Acad. Sci. 62, 681 (1969). c. B. Ward and D. A. Glaser, “Evidence for multiple growing points on the genome of rapidly growing E. coli B/r. Proc. Nat. Acad. Sci. 65, 800° (1909). . Cc. B. Ward and D. A. Glaser, "Analysis of the chloramphenicol sensitive and resistant steps in the initiation of DNA synthesis | in E. coli B/r. Proc, Nat. Acad. Sci. 6h, 905 (1969). (See beginning of list for more recent publications. > eared ae ee Se ee wm PR ge ee PeE OP LSE See wm 8 IT ma oi ¥ hy “AE se, BSE ‘ weg ad a te Soe nae a ee ate ey Bo. * ee FES Pas Ss * * a eH in.) ote te te etn aterm 6 me te me Nimo Om ; ee co eine a 2 ee ene Cogtvnuathe one 8 Donald A. Glaser eben Gam fe tapes - - BAT OEP aS TT __ Privileged Communication ' _ Eh LULULUCUCOCCtC‘S Ronalé Baler - Associate Development Ingineer. b. 2/16/29 in Fulhem, London, England. U. S. A. Ruislip Manor Secondary School, First Year Netional. Instrument Maker, Ingersole Ltd., England, 1951-54; Tool and Instrument Maker, G. E. ‘Research Labs., England, 1954-57; Mechanical Designer, Physics Department, U. of Michigan, 1957- 393 p Mechanica Designer. Lawrence Radiation Raboratorys.: 1959-65. John Bercovitz « Assistant Deve lopment maineer. b. 9/3 /k5 ; Baltinore, “Ma. Cal. Poly, Pomona, BSME, 1972. Design Engineer, Riverside 1969-1973. A a tern thes Mme ae OH James Berk - Associate Developzent Engineer, b. 9/17/42, Nev London, Wisconsin. UCLA, BA Physics, 1965; UCLA, MS Physics, 1967; UCLA, PhD Physics, 1969. ToC eT Biochemist, UcBerkeley, 1970-75. National Science Foundation Fellow 1966-57 . . UCLA, BA, 1957; UC Berkeley, MA, 1958; Teaching Assistant, Department of © Mathematics, UC Berkeley, 1939-613 Computer Programmer, Lawrence Radiation Laboratory, Livermore, 1961-65; Instructor, UC Extension Division, various semesters since 1961. _ John Couch - Research Associate. b. 5/6/h1, Hartford, Arkansas. MIT, SB Physics, 1963; Stanford, PhD Biophysics, 1970; Acting Instructor in Biophysics, St tanford, : 1971. {ss W. XR. Fair, J. L. Couch, N. Wehner, Biochemical Medicine 8 (329-339), Purifi- t cation and Assay o? the Prostatic Antibacterial Factor (PAP); Nakayama, H. and Couch, J. L., "Thymineless death in Escherichia coli in various assay ‘systems: viability determined in liquid medium", J. Bacteriol. 114, 228 (1975); Jd. I. Couch and P. C. Hanawalt, "DNA repair replication in temperature -sensitive DNA synthesis deficient bacteria", Biochem. Biophys. Res. Commun. 29, 779 (1967); J. L. Couch and P. C. Hanawalt, "Analysis of s-bromouracil distribution in | partially substituted deoxyribonucleic acids", Anal. Biochem. #1, 51 (1971); _ P. C. Hanavalt, D. F. Pettijohn, EB. C. Pauling, C. F. Brunk, D. W. Smith, L. C. Kanner, and J. L. Couch, "Repair replication of DNA in vivo," Cold Spring | Harbor Symposia, on Quantitative Biology, Vol. XXXIII (1968), Pp. 187. i | i | Fraser Bonnell - Principal Prograrmer. b. 7/28/35, Port Chester, N. Y., USA.. | j $ “Ted Fujita - As sistant Development Engineer. b. 9/19/43, Tovaz, Utah. Uc Berkeley, BS, 1964; UC Berkeley, MS, 1965. Project Engineer, Berkeley Selentific. Labs, , 1965-69. a ot — a ° ar we emma emo “Robert. ‘Henry. - Senior Develovinent Engineer. b. "6/8/36, Winfiela, ‘Kansas. U. Kansas, BS, 1959; UC Berkeley, MS, 1965; Boeing Airecraft-Electrical drafts man 1957-58 (summers) ; Western Electric Co-; Engineer, 1959-60; RCA, Engincer, 1960-64. | | Le Leif Hansen - Principal Development Rngineer. 5/29/27, Copenhagen, Denmart:. _ USA citizenship. ‘Technical University of Denmark MSME, 1954; Senior Design Engineer, General -Dynamics-Astr onautics,’ 1957-62; ‘Senior Engineer, Lawrence Radietion Laboratory, Berkeley, 1962-65. (vechanteak Eogineer, RDAP 1954-56). (cont inued), tote ‘Sp rom cht A vet os "s Bf 5 Po s af Poeeym RE Shwe BYE UN THIS “ = fs i ~~ 4 we, mop be . Coaatirantiog Tut oo” 9- Donald Ae ‘ Glaser. —— _ Privileged Communication ena “Larry Johnson - Associate Development Engineer. b. 2/28/37, Sioux Falls, South Dakota. §D School of Mines, BSME, 1959. Sperry, Project Engineer, 1959-65; Boeing, Design Engineer, 1965-66; FMC, Sr. Design Fngineer, 1966-70; - Thermidex, Sr. Project Engineer. Alex Para - Assistant Development Engineer. b. 2/22/50, Buenos Aires,. Argentine. Citizen of Argentina. Chabot College, AA, 19€8; UC Berkeley, BS, 1971; . ‘Engineer's Aid, UC Berkeley, 1969-70; Sr. Mngineers Aid, UC Berkeley, 1970-71. Sr. Developuent Ingineer, UC Berkeley 1971-72. co “oS i Wan os Donald.A.. Glaser. warner Sag2 ~ 9a = ~erivileged Communication... —_— _ Table of Contents Justification of First 12-Month Period Research Plan Specific Aims, Methods of Procedure and Significance Further Automation Instrumentation Development Significance Facilities Available , Collaborative Arrangements Principal Investigator Assurance 10 12. 2h 38 ho ho. Ta be -10- Glaser, Donald A. '. Privileged Communication Justification of first 12-month period Personnel During the first 12-month period we plan to maintain the engineering staffs at their present size, because we expect there to be extensive debugging, modification, and minor additions made to the machine as we gain experience in its use. Those who de- signed the machine will be the most effective at understanding its shortcomings and making necessary improvements. As time goes on the shop activities will shift from construction of new equipment to maintenance of the existing equipment at probably the same level of manpower as required during the construction phases of the project. The instrumentation and electrical engineering group will similarly be engaged in debugging, modifications, and minor additions to the equipment. , mo In order to operate the computer facility around the clock, we will need to have two full-time computer operators, but no other major expense is. contemplated. For biolo-~ gical operations, a Senior Biologist with considerable experience in computer program~- ming and instrumentation is being proposed and the budget also provides for the sala- ries of three postdoctoral researchers and four graduate students since training grants for these categories of people are no longer available. As the experimental program gains momentum, we will need to add two relatively junior programmers to help biologists formulate protocols and write programs to carry out the necessary operations. . Equipment a PDP1O-~I System to replace our PDP-6 System. By the time this proposed program begins in June 1975, we will have owned and operated: our present PDP-6 system for 1O years at an enormous saving in the cost of leasing the same equipment. Lease rates are usually computed to amortize the equipment in about 4O months and we will have operated the equipment for 120 months atthe same cost. Several years ago the PDP-6 computer became essentially obsolete when it was replaced by the PDP-10, and then by the PDPlO-I system. Probably by June 1975 there will be a yet newer replacement of the PDP1O-I system. At the present time’ (October 1973) there is only one operating PDP-6 computer left in the United States at the Rand Corpora- tion who are planning to get rid of it in the next few months. ‘There may also be another highly modified PDP-6 computer at M.I.T. not maintained by D.E.C. (Digi- tal Equipment Corporation) and perhaps used for special experimentation in compu-~ ter science. D.E.C. no longer maintains the software for the PDP-6 and it is costly and difficult for us to modify the constant improvements in PDP-10 soft- ware so they are useable on the PDP-6. New software, beginning to be issued by D.E.C., is not suitable at all for the PDP-6 computer and we will soon be unable to take advantage of the "community knowledge" and library of programs available for PDP-~1O applications. It is not practical for us to maintain the computer ourselves and D.E.C. maintains only one trained maintenance person who, in fact, can only be trained at our own computer by his immediate predecessor. We absolutely then depend on this one person because ours is the only computer of its kind still maintained by-D.E.C. The change to the new PDP 10 system most recently available in June 1975 is expected to give us a speed increase of at least a factor of h in analyzing photo- grephs from the Dusbwaiter and Cyclops. Since these instruments take photosranis at the rate of 1 per second and our precens rete of enalyzing pictures is about 1 psr 10 seconds to 1 pe» 20 seconds, we have an eutremely unfavorable ratio of analysis time to production time for these photographs. With this additional Glaser, Donald A. ~ll. a Se Privileged Commnication a Justification of first 12-month period (continued) factor of i or more available in the PDP-10 system, the ability of the computer to analyze. data will be nicely matched to the rate of production by the biological machines. For all these reasons, the switch to the new system is extremely desir- ab ie * . , . laser. This laser is needed to measure the light scattering of droplets of cell Culture formed in the high-speed dripper-inoculator in order to determine whether a droplet contains a cell and the kind of cell contained therein. By rejecting empty droplets and droplets containing multiple cells as described in the Biolo- gical Plans part of the proposal, we will increase the effective size of the Cyclops and Dumbwaiter by a factor of 3 and be able to carry out critical sorting operations for experiments on animal cells. Ss Flying-Snot Scanner--sp2ed un. To further increase the speed at which photographs can be analyzed, we propose to update the Flying Spot Scanner to current techno-. logy by the substitution of the PDP-ll computer to serve as a controller for the scanner in place of the home-made cireuit thet does the job now. In addition we will substitute new, improved versions of the deflection yoke system for the pre- cision cathode ray tube and faster A/D converters. Finally, we would add a PDP-~10 to PDP-11 direct memory access-dimp for bringing scanner information directly into the PDP-10 memory without going through the slower 1/0 Bus. Supplies--The cost of supplics is based on the assumption that the Cyclops will con- tinue to operate. for small-scale experiments and for "second-pass" experimental ‘material produced by lerge Duwsbwaitcr experiments. It will operate with petri dishes or with gloss treys at a modest level as described in the budget figures themselves. ' The budset for Dumbwaiter supplies is based on the expectation that we will be able to carry out 2 batches per veek for 50 weeks per year which seems at this time a reasonable average level of activity. . ~- Travel--On the average of one major trip to the East Coast for professional persons to attend a major conference such as the Gordon Conference and the Cold Spring Harbor Confermec, as well as attendance by professionals and graduate students at local conferences. Also conferences vith colleagues and equipment suppliers. Other--Computer maintenance contracts are based on present cost estimates by the manuiacturer who carrics out the maintenance. Machine-shop time and other campus shops is required from time to time vhen our own single machinist is overvorked or when special facilities and large machines are required for a particular job. The budget is baced on one man year of work for this purpose. ‘he Equipment Budget for subsequent years provides for new accessories bound to be required as the experimental program expands, including for instance, a television- scanner system for on-line real time analysis of growing colonies to eliminate the photography step and provide for the possibility of intervention in the experiment in real time and very rapid read-out necessary for particular applications. For study of animal cells it vill probably be necessary to design a camera that photographs a small area of agar at a time through a low-power microscope for studying very small clones of animal cells. Other requirements of these kinds are bound to arise. We will justify this budget item on a year-to-year basis. M emegmieatipes wed ae 1? ~ Donald A. Glaser we a . Privileged Communication 00 "Rescarch Plan AS insroduction | 1. Objectives. When this program-project began in July 1955, the overall goel wa: to automave many of the procedures of petri dish tecanicue on a large scale using computer-directed machinery and pattern recognition techniques in a flexible way so that a wide varicty of biomedical problems could be attacked. Now, in November | 1975, after successful operation of several prototypes, much of the equipment is in operation and all of the najor equipment will be in full operation at the end of the current grant period in June 1975. In its shorts period of operation the machinery has suecessfully aided in the ondole- tion of cold sensitive mutants of E. coli Kl2 unable to synthesize DNA at 20°C. Iz has also psrformed highly accurate automatic recognition of growing colonies of 10 species of bacterial pathogens important in medical diagnosis as.a demonstration of wr its abilities in many health-related applications. In the next few months we will 2 begin new experiments in genetic mapping, mutant isolation and- physiological charac- terization with H. coli, Saironclle typhimuriu:, Bacillus subtilis, Saccharomyces cerevisiae, and aninal cells grown in tissue culture. iiany os these projects will be Les done in collaboration with scientific investigators who have on-going projects in = these areas , . During ‘ che next five-year grant period beginning in June 1975, we propose to extend " these projects and add others involving the construction of genetic meps, the isola- i tion of important mutants, and the eharacterization of mutants and strains. Some , of these projects will be chosen to aid in critical steps of the productive work of i a& number of independent scientific investigators already working in these fields, rd and some will be important parts of our own biological programs. In addition, ve “fe propose to examine the feasibility of health related projects including screening x of environmental cheiicals, including food additives for their mutagenic effects on bacteria, ycast and animal cells, the potential carcinogenic and anti-neovlastic effects of various agents on animal cclls, and “the cffects of very low levels of Loniziny radiation on various cells. If large-scale screening projects appear feasi- ble and desirable, special funds will be sought to carry them out if necessary. Finally, a modest instruzentation program will be continued to add new capabilities to the machinery as they become necessery. Scientists from many laboratories are expected to take advantage of this facility. oo fee 2. Backsround. Since this progran includes a number of different biological es projects, the vwiological background, rationale, aims, and methods will be discussed a project by project in subsequent sections of this proposal. What brings them toge- ry: ther in this program is a similarity in the technical manipulations and the common Ss - requirement for large-scale experiments too tedious, slow and costly to carry cut by hand. In some cases quantitative measurements on proving colonies are extremely difficult without the automatic pattern recognition facility. Except for cnall labor-saving devices, techniques for growing colonies on solid media have changed little sine they were invented. Many projects in the contempor- ary biology of clonable cells are severely limited by the difficulty of isolating particular mutants, characterizing them, and locating them on the genetic hap of the organism. Numerous health-related programs including medical bacteriology; contami- nation monitoring; mass screening programs for mutagens, carcinogens, and anti- “neoplastic agents; and industrial strain-improvement programs utilize simile r teche~ nigues. It is hoprtd that this orocrom-projicet will be useful in all these fields ac wall as in vork in Vundenontel biolocy. fe mete Homme i a t 4 * Ree Se eR A GR MUR = em ne Gat | i t Donald A. Glaser rr Conteugntion rage - 13 - Privileged Comunication 7 nee 3. Rationale of this automation. To carry cut experiments requiring study of large numbers of colonics we are constructing a machine (the "Dumbwaiter") in which .256 hOcem x 80cm eagar-filled glass trays circulate in an.incubator past stations where | various operations can be performed. An inoculation device deposits single cells .carried in microdroplets in regular rovs and columns for maximum uniform packing and- easy subsequent manipulation. During incubation, time-lapse photographs of the colo- nies are made using up to 5 different colors of light. A flying-spot scanner. (similar to a television camera) under control of a computer (PDP-6) examines the photographs, finds all the colonics, and records their size, appearance, and growth rate. The. computer then computes the frequency of various clesses of colonies for measuring. muta~ tion rates, map distances, recombination rrequencies, and other required biological results. In addition the computer can direct a colony "picker" to retrieve part of a colony for replica plating, sus spension in liquid, restreaking, or delivery to a test tube or small petri‘dish for further manual work in the laboratory. Alternatively the computer can direct the spraying of some or all colonies with nutrients or drugs on some predetermined schedule or according to the actual performance of each parti-~ cular colony. Thus the conputer ean intervene in on-going experiments. Irradiation, genetic crossing on the agar, and similar operations can also be performed as the trays move through the Dumbwaiter. Design of the DW (Dunbwaiter) and associated equip. ment has been done to allow a wide variety of accessories to be added to carry out. epectal manipulations as they are required for particular experiments. If colonies ze placed 1 mm apart, the DW can hold almost 10% colonics per load of 256 trays. | Several loads can be processed each day for many types of non-~interfering experiments. * } What does this kind of large-scale automation have to contribute to -biomedical science? ; Solution of many biological problems depends’on the. ebility to isolate a particular ‘ kind of mutant, to measure the rate of a particular genetic recombinational event, or to measure responses of growing cells to specific chemical, biological and physical _ conditions. Automation allows highly reproducible experiments to be performed with large numbers of organisms so rare events can be observed and more common evénts meas- ured with high statistical accuracy. Computer-directed pattern recognition allows quantitative aspects of growth to be explored for regularities that would escape _ quest tative visual examination. , None of this increased statistical and quantitative power reduces the neéd for thought- ful study of the biological system in advance of large-scale experiments and of careful analycis of the results. Nor is this kind of automation likely to reduce the number or quality of people involved in a given research area. ther the same people will be able to accomplish tasks impossible without the machinery and to do many more- con=- ventional experinents with much reduced tedium. ! In medica 1, public health, and industrial applications, large scale sereening, con- | tamination surveys and diagnostic assays, and other similar tasks can be done with | : : nem ern Oe mi eC the unbiased reliability of automation and the cconomies of large-scale. It is expec- ted that these machines or adaptations of. them will be cost-effective and quality- leffective for a variety of immediate health-related applications. ~ —h. Comprehensive Progress Report. (a) Period covered by this report: June 1970 to November 1973. (b) Sumary. Equipment has been ‘built for inoculating up to 100 40 cm x 80 em agar-filled glass treys.with sins “le cells in regular rovs and columns of adjustable epacing, incubatins the treys under timttly controlled conditions, photographing them puriecdically, and analyzinh the photocraph: with e seamer-conouter system. Frecuen- cles of various colony types are recorded by the computer which can also direct the Donald A. Glaser Coatinuntios rode « Uh z ‘Privileged Communication ss automatic picking, replica plating, and restreaking. of colonies it is instructed to select. Nutrients, drugs, viruses, and other agents can be delivered to whole trays or selected colonies under computer control. Design and construction of a fully-automated system able to carry 10° colonies on 256 trays is near completion. . With the. presently operating system ve have isolated cold-sensitivé mutants of E. coli Kl2 unable to synthesize DNA at 20°C using 1/5 as much agar and much less labor than a parallel project using hand methods.. Nine bacterial species isolated from human urine and a laboretory strain have been studied with the automated system. Using newly developed programs, the computer can correctly identify unknown colonies of these ten types with accuracies better than 98%. m, A. 4(c) Detailed Report 1) Biological Projects. Although the goals and budget of this program-project were directed principally tovard development of the automation system, several biomedical projects have been carricd out to demonstrate the abilities of the system and to speed the work on biological projects in our laboratory supoorted by NIH as GM 19439 - (replacing GM 12524). The NIH preferred funding the instrumentation and biology pre- grams separately so that they would pe reviewed separately by appropriate panels. (a) Finding, Counting and Sizing Colonies. Computer programs have been written for scanning photographs of 1OO-mm-netri dishes prepared by hand for finding, ’ counting, and sizing the colonies correctly,in spite of overlaps of colonies ~ and wide variation in colony sizes. The counting algorithm has an accuracy of better than 99% on dishes containing up to about 400 colonies and’ requires about 10 seconds per dish. It is-thus greatly supericr to any commercial colony counter, but is not used currently because simpler and faster program’ are effec- tive with. regular array-inoculated dishes and trays. - (b) Isolation of Cold-Sensitive Mutants’ (by a method widely applicable to mutans hunting). Invectigationus of DNA synthesis in E. coli, its control, and its connection with cell division, require isolation and genetic mapping of conditionally lethal DNA mutants. Work on our laboratory and many others has uncovered 7 or 8 classes of heat sensitive mutants normal. at 50°C or 37°C but ‘unable to synthesize DNA at about h1°c. ‘These classes map at 7 or 8 distinct ~* sites, but probably DNA synthesis is even more complex and additional sites — defining more structural or control genes remain to be discovered. We are searching for new classes among coldesensitive mutants unable to synthesize DNA at 20°C by taking time lapse photographs of colonies grown from mutagenized cul- tures and shifted between 20°C (restrictive temperature)‘ and 37°C (permissive temperature). Imposing conditions on colony diameters and growth rates leads to efficient selection of cold-sensitive mutants by the scanner in a vay that saves much labor and materials when compared with a competitive hand experiment run in our laboratory. Cold sensitive mutants mapping at a known site (Cc class) have been found and 3 new mutants may represent a new class not yet precisely mapped. Since the colony picker is not yet in operation, the scanner aids in locating mutant colonies by displaying a map of each dish on the display scope. Holding the dish egainst the screen, mutant colonies are picked wherever the commer has drawn an kK. _systen semi-automatically on a reduced scale (about 100 trays maximum capacity). ‘ Photographs are examined by a flying-spot scanner (similar in operation toa tele- Copiaut tion octee 15 & | . Donald. A. Glaser ome og Privileged Communication _ _— This method (later -using tne picker Lo“eliminate hand labor) can ve used for any mutant selection based on colony size or appearance. we have tested it success- fully with knorm leucine auxotrophs by growing mixed prototrophic and auxotrophic . eultures in Limiting leucine and then spraying the agar with additional leucine, . taking photographs during the incubation intervals. 7 . (c) Automated Recopnition of Bacteriel Strains by Analysis of Colony Morvholory. To test the ability of the system to identity bacterial pathogens for medical and public\health applications, we photogrephed 24-hour colonies of nine species isolated from human urinary infections plus one Bacillus subtilis strain. Using methods of colony morphology analysis dcecribed below, tne system “learned” to -recognize the 10 test species by examining about 1000 colonies of each. Upon scanning an additional L000 colonies presented in mixcures or in pure cultures, the program makés tvo decisions: 1) whether to attempt an identification (ans- wered "no" if the "colony" is not round, is actually a piece of dirt, an imperfec- tion in the agar, etc.), and 2) to what species does the colony belong (if 1) is. ansvered "yes"). Results were as follows: . * . % Attempted _ % Correct Aerobacter aerogenes . 83 ~ * 200 Bacillus subtilis - 8B 100 Escherichia coli , 83 , 100 Herellca vaginicola TT 100 Klebsiella pncwnoniae 81 - Proteus morganii 86 - 00 Pseudomonas putida . 83 - 100 Salmonella typhimurium . 89 . ,100 Serratia marcescens 86 100 Staphylococcus aureus 89 > 100 2) Technical Prosress. When completed in January 1975, our automated system will prepare minimal agar medium in 400-liter batches, dispense it with a prograrmable variety of additives into 256 hOcm x 80cm prestcrilized glass trays, and circulate. “the trays inside a precision incubator past stations for inoculation, time-lapse photography, colony picking and replica plating or restreaking, and treatment with chemicals, radiation, viruses, etc. In January 1975, the prototype test version came into operation and is nov carrying out almost all of the operations of the final ty vision camera) connected to a medium-sized computer. The computer finds all the colonies, measures their diameter, characterizes their appearance (using up to about’ 100 parameters), and issues commands for colony picking, nutrient spray, mxtant ‘purification by colony restreaking and replica plating, according to a protocol written by a biologist. : . By June 1975, when the presently proposed program is due to begin, the system should be in full operation. Technical aspects of this vork have been reported in two published papers: D. A. Glaser and W. H. Wattenburg, An automated systemtor the growth and analysis of large numbers of bacterial colonies using an environmental chamber and.a computer-controlled flying-spot scanner, Ann. N. Y. Acad. Sci. 139, 243 (1966); D. A. Glaser and C. B. Ward, Computer identification of bacteria by colony rorpholosy, Frontiers of Pattern Recognition, Academic Press, N. Y. (1972), - : ~ ¢,;™ SEIT Fey 2 eat r : Se Be ye ae oa Centinestias sme 16 0... _ Donald A. Glaser. _ Privileged Comaunication a Nee ee oe ese vente eR oe ee me eee te me me OL Fa eel aR ett can no mmr Mtl ne anes trae ane tne mmm oe a larg re number of oral reports, and Progress Reports to the TICMS. Detailed publications will be: ‘prepared after the full system is completely operational. -We ea seen no reports of sinilar systems in operation elsewhere. — . (a) Oo jectives and General ‘Description. Many of the biological opdect ives of this program require the ability to examine about 10° fairly well isolated,colo- nies (about one colony per square centimeter). For other studies up to 108 colonies need to be examined but they may be crowded into a smaller space (about 100 colonies beg square centimeter). The machine must therefore have a capacity of about 10° ¢ em’ of solid grovth medium (agar, silica gel, or other medium) s To - provide the required area of agar in the smallest possible volume, the machine uses stacks of horigontal agar-covered trays spaced one inch apart. These trays are made of inespensive window glass with metal frames and can be washed and serilized very easily by reasonably standar techniques. They elso provide a very uniform growth surface of high optical quality so that good photograplis of growing colonies can be made. Ordinary plestic or glass pnetri dishes made by hand in the leboratory in small batches in the conventional way can be laid on the trays for incubation, photosraphy, and manipulation in the machine. Al- ternative ly, Large-scale e3 ~perinents ean be carried out by pouring a sheet of agar directly on the tray. A design has been chosen which makes it possible to intermix these two modes so that the petri dishes made by hand can be analyze at the samc time as large-scale experiments prepared automatically by the machines The entire machine is fully automated to perform large-scale microviological ex perinents in conjunction with o sophisticated data gathering and processing system. Because the stacks of trays are moved up and down by. mechanical devices, we have called the machine “A Dumbwaiter" The design concept of the Dumbwaiter is very simole. Glass trays carried in aluninun frames are stacked directly on top of each other in two stacks about 25* apart. Cross-ducts are“provided to transfer trays from the top of one stack to the top of the other, and from the bottom of one stack to the bottom of the other. The trays then circulate in a rectangular path moving up through one stack across to the top of the other, down through the second stack, and across from the bottom of the second stack back to the bottom of the first stack. This over-all design plan can be seen in the attached figure. On tl cross- ducts for moving the trays horizontally will be mounted cameras for photograph- ing the trays and special aceessores for inoculating the agar with organiems; administering drugs and nutrients, irradiating with ultra-violet light or other | radiation, picking, restreaking and replica plating colonies and other manipu- | lations. Trays are handled singly only in the cross-ducts. In every other part of the Dumbwaiter and auxiliary equipment, the trays will be handled in stacks of 64. The stacks and transfer paths are enclosed in housings in which a sterile grovth, environment is acntained. Mixing, sterilization, and pouring of agar is carried out outside of the Dumb- waiter. Accessory stations will also be provided for washing the trays for reeuse, for sterilizing them, and for special incubation and cold-storase of trays of colonies which do not need to be vhotosraphed very frequently. Four moveable magazines will be provided for storing stacks of trays and transporting them from the Dunbwaiter to and from various auxiliary stations where these special operations will be carried out. The separation of these necessary func- tions to a number of specialized stations was phehaia to be the best vay to provide : , . 2 renid, reliable ond econonica), oneration of the svetem. On the following pares ‘uve WLLL give the deteaile, status, and churachariesies oP tne Diesbusiter and its rd cg ae Sep gem an f . aunaliar; COUT Ge f A wee 4 & 3 OTS eo pad tel A Res SFT yey ae gta i we oon nae ele ¥ m ete 7 tat tat. RAL Oath (S OA bea VI a WOie Loanitnuctige C290 17 - Donald A. Glaser privileged Communication. ..._ €i ma (b) Operational prototype (cyclops) + As design. aill.testing of Dumbwaiter com- ponents procceds, we often need to construct temporary devices for testing de- sign principles and mechanical devices that will be used in the Dmbwaiter. At the same time, we were anxious to begin carrying out biological experiments before the Danbwaiter comes into full operation. We have, therefore, constructed a machine celled "Cyclops" consisting of a Dumbuaiter camera mounted on an x-y motion capable of handling one or tvo Dumbwaiter trays in the same way that vill be..done in one of the horizontal cross-ducts of the completed Dumbwaiter. Cyclops is capable of photographing agar-laden glass trays or trays carrying conventional plestic petri dishes, of inoculating sterile agar with organisms to be grown). of spraying drugs, nutrients and other substances, .of picking and restreaking colonies and carrying out most of the mechanical and optical operations of the Dumbvaiter.. It is not. able to incubate and circulate trays, however, end at the present time vequires the trays to be transported by hand. Nearly all of the. other ancillary facilities of the Dubwaiter are being used routinely for experi -, ments done on the Cyclops as will be described below. (c) “Moveable macazines. The moveable magazines serve many purposes. Their main function is to sranspors and protect the 6l-tray stacks. Each stack rests on a dolly on rails on the bottom of this magazine. When the moveable magazine is engaged to a fixed magazine for transferring a stack in or owt of the Dumbve iter, the rails in the moveable magazine mate with corresponding rails in the fixed magazine. The moveable megazine and the fixed magazine both have doors facing each other. ‘The space left between the doors after engagement is accomplished Will be sterilized by UV radiation: The doors vill then ve coupled together ‘and simultancously litted up into an enclosed UV irradiated container above the fixed magazine. The lifting of the doors is performed py an air cylinder. The ‘stack transfer can now be executed using a hand-driven transport serew located in the moveable magazine. - ‘Whenever actual stack transfer is not taking place, the dolly is locked in a fixed horizontal position and the stack is secured in vertical compression by hand-operated screws in the magazine top cover. This will prevent unwanted move- ment of trays in the stack during transport and handling of the magazine. The vertical compression will be especially important to keep all trays paraliel to cach other during the agar-pouring and annealing process. The agar-pouring will be done while the moveable magazine is reeting on levelling jacks in a combination sterilization, pouring, and annealing oven. ‘The stack dolly is > equipped with mercury levels (permitting 180°C dry sterilizat ion) to assure accurate Levelling of the stack before the agar pouring. ‘lhe agar Vi: rology 45, 366 (1970). 2. M. Hane, "Some effects of nalidixic acid on conjugation in Eecherichia coli K12", Je Bact. 105, 45-56 (1971). —_s- rs 3, C. Be Ward and D. A. Glaser, "Inhibit tion ‘of initiation of DNA synthes is by low - concentrations of penicillin" ; he RM. Burger, "Kinetics of labeling of fas b-renaturing WA in Bacillus subtilis", J. Mol. Biol. 55, 199- 201. “(a9n). ‘ 7 - RR. M. Burrer, Mmolucnesiented Reoherichta oo replicate only that DNA which iW. Aead. Set. 68, flo% (1971). ryt age oe + + yt — was aboub to ne renlicetcd inv ag al tain a a Cantippesss ae «Be Donald A, Glaser. _Privileg ged Communication oo . ne 6 R. HN. Burger and D. A. Glaser, "Effect of nalidixic acid on DNA replication by tolucne-treated Escherichia coli", Proc. Nat. Acad. Sci. 70, 1955 (1973). Te D. Le Parker and D. A. Glaser, "Chromosonal sites of DiA-nembrane attachment in Escherichia coli" ;» submitted to J. Mol. Biol. September 1973. -8. D. Le Parker and D. A. Glaser, "Effect of growth conditions in DNA-rembrane attachment in Escherichia coli," in preparation. -o 9. A. H Dougen and D. A. Glaser, "Rates of chain elongation of ribosomal’. ‘RNA molecules in Escherichia coli", submitted to J. Mol. Biol. 1973. 10. L. Waskell and D. A. Glaser, "me isolation end partial characterization of mutants of E. coli vith cold-sensitive synthesis of DNA", in preparation. wed ae vf ? a Wes vat A. he. Staffing Assistant Professor of Clinical Pathology 1970-71 W. Keith Hadley 7 and Laboratory Medicine, UC Medical Center. Postdoctoral Fellow 1967-69 oa a ~ Leif Hansen larry Johnson Alex Para Principal Development Engrg. Associate Development Imergc. Assistant Development Engrg. Calvin Ward - ‘ Assistant Research Biologist 1969-71 ant -Beverly Wolf Assistant Research Biologist 1965-72 a Ronald Baker Associate Development: Encre. 196h-present m. John Bercovitz Assistant Development Ingreg. 2/73-present te James Berk Associate: Development Iberg. 3/3 ~present ed Fraser Bonnell Principal Programmer - 1965=present ef John Couch Research Associate - 1971-present be fed Fujita Assistant Development Engrg. 1969-present aN Robert Henry Senior Development Engrg. 1964-present 1965-present 2/73-present 9/T2-present ~ oh . Glaser, Donald A. Privileged Communi cation 2a B) Specific Aims; C) Methods of Procedure; and D) Significance Since this program project is a collection of different biological projects, we will devote a section of this proposal to the aims, methods, and significance of each. project separately. These projects have in common the need to isolate and char- - acterize mutants or recombinants difficult to find by hand methods because they are rare and have no easy biological or chemical selection technique, but can be defined by growth rate or colonial morphology under particular growth conditions. In some cases the events are not rare but their frequency must be known with high accuracy so that large numbers of colonies must be examined. The isolation procedures involve | inoculation with single cells, incubation, time-lapse photography, replica plating, colony picking, colony restreaking, growth rate or morphology analysis, and other Operations -that our system is designed to carry out on a large seale. Some of these projects are already under way; some will be begun soon; others will require prelimi- nary feasibility studies; and still others will be added later. ‘They represent a sampling of projects proposed in conversations with a number of scientists and involve @ range of clonable cells from bacteria to mammalian cells. They include fundamental studies of molecular evolution and biochemical pathways as well as applied studies of mutagenic effects of environmental chemicals and efficacy of proposed antineo- plastic agents. With each project title is listed the scientific investigator(s) who propesed and will guide the work. In some cases a true collaboration with our laboratory is expected to develop; in others the effort will be to help provide mutants for independent and on-going research done in other laboratories; in still others a feasibility study or actual screening effort with direct health-related goals will be undertaken. 1) Isolate, map, and characterize temperature sensitive mutants of E, coli unable to synthesize DNA at 20°C or at 41°. oo Donald A. Glaser, Professor of Physics and Molecular Biology, University of California, Berkeley. “Method: . Automated replica plating and incubation at the permissive and restric-. tive temperatures followed by photography and computer matching of replicas is a straightforward method that will soon be possible. In current use is a series of time-lapse photographs taken of single primary colonies incubated at permis- sive, non-permissive, and permissive temperatures on a time schedule that allows the computer to impose limits on the colony size to define the mutant class. selected. Less agar and fewer manipulations are required for the time-lapse method, but some mutants may be killed at the restrictive temperature so different classes of mutants may be produced by the two methods. Mapping is done by inter- rupted mating or epvisomal complementation followed by measurement of co-transduc-~ tion frequency. Results are obtained by automated colony counting on selective media. Characterization of mutants will be done mainly by conventional methods. Significance: Knowing the number and location of genes involved in DNA synthesis and its initiation in E. coli is the first step in the genetic and biochemical dissection of this all-important cellular process. Mutants obtained in this study will be shared with other laboratories engaged in enzymological analysis to speed the overall progress in understanding DNA synthesis. (Dr. William Wickner in Professor Arthur Kornberg's laboratory, Biochemistry Department, Stanford University, is studying one of our cold-sensitive mutants that may repre- sent.a new DNA gene). An understanding of this most complex and central. process in bocteria is bound to be imnortams for understanding the analogous processes in cells of higucr organisins, including. proliferating animal ceils. Alternatively antibiotics that function by perturbing DNA synthesis may be understood or Glaser, Donald A. Privileged Communication 2a. 2) 3) rationally sought if vulnerable features of DNA syfithésis in pathogens is understood. feasure anomalous DNA synthesis events. for temperature-sensitive mutants, for UW sensitive and UV resistant mutants, and for recombination~deficient mutants, including gene duplications, deletions, point mutations, other chromosomal changes. D. A. Glaser Methods: Changes in proteins involved in DNA synthesis may produce detectable changes in the rates of occurrence of various mutational events including point mutations, deletions, and duplications. The rate of point mutations can be estimated from the rate of revertable auxotrophs. Deletions can be scored as non-reverting auxotrophs, and duplications can be ‘scored by assays for certain enzymes. In particular colonies are able to grow on lactobionate as sole carbon source only if there is a duplication in the lactose operon. Chlorate resistance is being used as a selective condition for deletion of chlorate genes whenever a nearby site for some other function is also affected. These and other assays will be used to study the roles of various DNA synthesis-related genes known to affect UW sensitivity, recombination, or any of the genetically-defined class of temperature sensitive DNA mutants, whether enzymatically characterized or not. Significance: In evolutionary changes to optimize survival, certain changes in the chromosome must be advantageous in pruning away unnecessary DNA, duplicating genes required to produce large amounts of product, providing surplus duplicate genes for future mutational experiments, and enlarging the chromosome to provide scope for greater complexity. The probability of these changes must be affected by the structure of DNA synthesis-related proteins. An understanding of these effects is critical for understanding evolution at the chromosome level and also necessary to understand diseases of higher animals that may result from slight perturbations of the DNA synthesizing machinery. Rational searches for anti- biotics against bacterial pathogens may be possible if this class of perturbations in their DNA synthesis can be understood. Intensive mapping of the E. coli chromosome and measurement of changes in size of the chromosome as frequencies of various mutational events are changed. D. A. Glaser Methods: i. Temperature sensitive lesions can be. readily introduced into the bacterial chromosome and mapped by Pl transduction using the already fairly densely placed well established markers in E. coli (or using P22 in Salmonella). Thus the map can be densely filled with temperature sensitive relatively well localized mutants. ; ii. Temperature sensitive mutants which densely cover small local regions of the map can be prepared by mutagenizing P22. transducing phage in Salmonella’ (or Pl transducing phaze in E. coli) and transducing in a wild type gene for a known lesion on the recipient strain. Transductants for this particular marker gene will then carry a number of lesions in the neighboring region (around 1% of the chromosome) corresponding to the size of the transducing phage (this method has been develoned very succes sfully in recent work (Hong, J., Smith, G., and Anes, B. W., FEAS 23, 0899 (1971))._ - 26 Glaser, Donald A. Privileged Communication , fs iii. Ordering of mutants within the close neighborhood of Bich ‘other can be done by two and three factor crosses by generalized transduction and also by a new episome complementation method developed and described by Robert N. Reeves and John R. Roth, JMB 56, 523 (1971). Use of automatic techniques will allow the enormous labor required to make an intensive map to be done easily using transduction, mating, and other techniques that can be carried out on agar. The establishment of a large library of temperature sensitive and more completely characterized mutants covering the chromosome map thoroughly would have very many applications in the study of bacteria and especially of yeasts and higher organisms. We propose to begin the work with bacteria for which the techniques seem straightforward and extend it later to higher organisms. Significance: i. By periodic measurement of map distances by cotransduction or interrupted mating one can monitor increases and reductions of the chromosome by the net effect of gene doubling, recombination, deletion and other processes that may affect its size. With a large number of standard markers and standard proce- dures the machine can keep a steady picture of the state of integration or autonomy of various plasmids, of the chromosome number, if that is subject to change, and of the size of the chromosome... It seems more. likely that the size of the chromosome is not an accurately conserved quantity but there will be variations in the population and it is hoped that methods of measurement will be sensitive cnough to make some description of this distribution and how it changes when the parent strain of the population contains various mutations especially affecting DA replication and repair. , ii. There may be regions of the map for which no temperature sensitive mutants or other conditicnally lethals can be found., It is of great interest to know how much of the DNA specifies no function and is functionless except for its role in evolution of new genes to carry out new functions or for structural functions at the DNA or RNA level. iii. When the whole map or at least regions of it are densely filled with markers it may be possible to discern overall patterns of placement and organi- zation of the genes according to their function or evolutionary history and thus to understand better evolutionary or physiological demands that led to this particular pattern or structure. The operon concept is the most obviously important fact of this type but there may be peties as yet unrecognized. iv. It will be possible toa supply large numbers of densely located temperature sensitive anu other kinds of mutants in particular regions of the map for inten- sive further study of particular problems in this and other laboratories. We intend to use the method immediately for trying to generate large numbers of mutants in tne neighborhood of known sites for DNA regulatory mutations hoping to discover other DNA regulatory mutants in the same neighborhood. As the techniques develop we will probably be able to supply other laboratories with large nunbers of mutants important to their particular interests. If eutomation makes it possible to follow changes in a densely-mapped bacterial chromosome subjected to a variety of mutational situations, the resul- ting insipht: into chrenosene mechantes will be extremely va tuapics axtension Gv Ghis appreneh to ehiuccosa: ml dpbrner i her irmortene conecaucnecs Vor witereiandine a vide rane oe eilitdes pacvunetoaac gor cecil in which * good biological mechanisms of genctic recombination are available. _ 27 « Glaser, Donald A. Privileged Cormunication * , 4) Genetic characterization of the chromosome terminus and the regulation of cell division in E. coli. , David R. Zisman, Assistant Professor of Bacteriology, University of California, Berkeley. Ss Methods: Recent evidence from gene frequency measurements (1-3) autoradiography (4) and biochemical analysis (5) demonstrate bidirectional chromosome replication in E. coli. The origin of replication appears to map at about 75 map min while the terminus has been mapped at about 30 min (6). The termination of chromosome replication appears to be necessary for chromosome segregation and subsequent septum cross wall formation (7-9). - . It has been suggested that chromosome termination may trigger division by the - transcription of division related genes, located at the chromosome terminus, at the time of their replication.(7,10-15). This hypothesis has recently received some experimental support: (a) studies of cell division following DNA, RNA, and protein inhibition at the time of chromosome termination in synchronous cultures (16-18) in@icate that the specific replication of the lest 0.5% of the chromosome. (0.45 map min) is required for subsequent cell division; blocking protein synthesis during this replication will block the subsequent cell division. (b) Several filament forming septation mutants have been obtained which map near 30 min, the chromosome terminus (15, 19-20). : Unfortunately, the region of the genetic map around 30 min is one of the most poorly understood areas (21). Very few markers have been identified; a stable F' has never been isolated for this region (22). We therefore propose to study this region of the E. coli map in great detail using the automated techniques now available. Hopefully the study of this region will help us understand the nature of the link between chromosome termination and cell division. We have isolated a man~ mutant (30.5 map units) that is non-reverting. We propose to use the transducing phage P, to cotransduce mutagenized markers (23) from a mant strain to our man~ strain. Transductants grown on mannose minimal medium will be plated out using Dr. Glaser's automation equipment, replica plated at different temperatures, and temperature sensitive colonies obtained. These colonies will be characterized for nutritional defects or division defects. The nutritional mutants will be saved to help us map this region of the chromosome. The division-membrane mutants will be studied more carefully to determine possible relationships with chromosome structure and/or regulation of division. Complementation studies should indicate the specific number of division related genes localized in this region of the chromosome and the possible existence of a division operon. Double mutants will be prepared so that the in vivo interaction (epistasis) of known mutants of different phenotypes can be studied (15). This approach can lead to the sequencing of related gene functions and is the first step necessary to determine the ordered pathway for septation in a manner similar to the study of T-even phage development and other self assembly systems (2). i. Masters, M., Proc. Nat. Acad. Sed. U. S., 65, 601 (1970). 2 ‘ ‘ey aves £5c, 1b? (1971). ? ? o 2. Bird, 2. B., Lovern, J., Martuscelli, d., and Caro, L. G., J. Moi. Biol., 70, 549 (ly72). | Mey ek says . eye ue aa Se ROSverS, He, Od Loode, F. 28 - Denald A. Glaser Privileged Communication a! 20. 2c. 23. 24. —§) Prescott, D. M., and Kuempel, P. L., Proc. Nat. Acad. Sci. U. S., 69, 2482 (1972). McKenna, W. G., and Masters, M., Nature few Biolozy 240, 536 (1972). Hohlfeld, R., and Vielmetter, W., Nature Tew Blolozy 242, 130 (1973). Clark, D. J-, Cold Spr. Harb. Symp. Quant. Biol., 35, 823 (1968). Helmstetter, C. #., and Pierucci, 0., J. Bacteriol., 95, 1627 (1968). Walker, J. R., and Pardee, A. B., J. Bactcriol., 95, 125 (1968). Clark, D. J., J. Bacteriol., 96, 121% (IS68)._ Hirota, Y. A., Jacob, F., Ryter, A., Buttin, G., and Nakai, T., J. Mol. Bioi., 35, 175 (1968). | : Pierucci, 0., and Helmstetter, C. E., Fed. Proc., 28; 1755 (1969). Previc, E., and Richardson, S., J. Bacteriol., 97, 416 (1969). Pritchard, R. H., Barth, P. T., and Collins, J., Symp. Soc. Gen. Microbiol., 19, 263 (1969). ' ' Zusman, D. R., Inouye, M., and Pardee, A. B., J. Mol. Biol., 69, 119 (1972). Jones, N. C., and Donachie, W. D., Nature New Biolozy, 245, 100 (1973). Dix, D. E., and Helmstetter, C. E., J. Bacteriol., 115, 786 (1973). Marunouchi, T., and Messer, W., J. Mol. Biol., 78, 211 (1973). Hirota, Y., Ricard, M., and Shapiro, B. In L. A. Manson (ed.), Biomembranes, Vol. 2, Plenum Publishing Co., New Yors, pp. 13-31 (1971). Ricard, M., and Hirota, Y., J. Bacteriol., 116, 314 (1973). Taylor, A. L., and Trotter, C. D., Bacteriol. Rev., 36, 504 (1972). Low, K. B., Bacteriol. Rev., 36, SO7 (1972). Hong, J., Smith, G., and Ames, B. N., Proc. Nat. Acad. Sei. U. S. 68, 2258 (1971). Eiserling, F. A., and Dickson, R. C., Ann. Rev. Biochem., 41, 467 (1972). Significance: Detailed understanding of the relationship between DNA synthesis and cell division in E. coli may give important insights into the same relation- ship for proliferating animal cclls, which generally do-not synthesize DNA except in preparation for cell division. som Studies in biochemical evolution in E. coli and B. subtilis. Joshua Lederberg, Professor of Genetics anc Biology and Cnairman of the Genetics Department, School of Medicine, Stanford University, Stanford, California. We wish to observe alterations in polypeptide products resulting from mutations in synthetic genes (generally synthetic horopolymer sequences) which have been inserted into the genomes of E. coli and B. subtilis bacteria. Immunochemical methods will be used for detecting these alterations by examining large numbers of small colonies for which no biological selection condition is known. By observing evolution of a polypeptide, much can be learned about the genetic code and about rates of various kinds of mutations in different nucleotide environments. Genetic organization of the E. coli chromosome: mutation rate versus map position of the translocated lactose operon. Gordon Edlin, Associate Professor of Genetics, University of California, Davis, California. The purpose of these experiments is to probe the genetic organization of the FE. coli chromosome. Ultimately we would like to understand why genes are located at particular sites in the chromosome. One approach to this question is to measure frequency of mutations in a gene (or genes) which have been translocated co nudr of divverony sives in the ehvemusen:. A model syste. for theee ex- periments is provided br the luesase oneron. A set of stroins exist vhich are genetically unifora except that the lactose genes have been translocated to a Privileged Communication . — Wii Ce 7) ~29- Donald A. Glaser number of different sites in the chromosome. These strains will be mutagenized with a varicty of mutagens (nitrosoguanidine, ethylmethane sulfonate, U.V. light, etc.) and the frequency of lac’ — lac” cells will be measured. Preliminary studies have shown that the frequency of mutations in the lactose genes are a function of chromosomal location. After analysis of the lactose genes, the same analysis can be applied to other genetic systems such as an amino acid biosynthetic pathyvay, ribosomal protein, etc. Genetic techniques for constructing the appropriate bacterial strains already exist. WeEnclosed is.a brief statement for your grant. We would like to go ahead on this as soon as possible since it is all worked out and is basic- ally a matter of cranking out the. data. The diagram shows the nine strains we want to test. The lac genes are located at the 9 positions wv hee 7 ne we an ee e . We will mutagenize with ENS wand nitvosoguantdine for starters. We can measure the mutagenesis here by measuring the number of valine resistant colonies. That gives us a number to use to normalize the mutagenic effect- iveness. We would then bring down the mutagenized culture to be sprayed onto trays. We want to test the number of lac” cells. I think the easiest way to do this is to place them on EMB lactose agar. Lac? are red and Lac” are white. We probably need to photograph at 2 or 3 times to reliably distinguish the 2 types and probably have to set some limits in the computer as to what it calls white and what it calls red so we probably need a dry run. Once that is determined we can run them as fast as time allows. I presume we will work with Phil on this. Let me know how and when you want to proceed. “ Recombination deficient mutants of E. coli. . moe Alvin J. Clark, Professor of Molecular Biology and Bacteriology and Immunology, University of California, Berkeley. Method: "Our worl in large measure stems from the discovery of recombination deficient mutants of various recombination preficient strains of E. coli. In doing the necessary mutant hunts the present bottle-neck is the picking of colonies of survivors of mutagenic treatment and patching them in geometric array. I am very interested in testing the dripper you have invented as a means of depositing cells in ogonetric arrey prior to testing their clones for recombina- tion deficieney. Is te very noocible this isc feellitete meny Capea net s we have 2 ° 2 n 3 been sitting on because of the Lasor involved in piching and patchinc. 9) 10) 1) Donald A. Glaser Privileged Communication gy i ° ~ New Salmonclla typhinuriua tester strains for cetecting mutagens and carcinogens among environmental chemicals. Bruce Ames, Professor of Biochemistry, University of California, Berkeley Method: This work is an extension of work already published to special cases for which the labor of mutant isolation and characterization is Limiting. i. ‘Ames, N. B., Lee, F. D., and Durston, W. E., Proc. Nat. Acad. Sci. U. S., 70, 782 (1975). 2. Ames, N. B., Durston,W. E., Yamasaki, E., and Lee, F. D., Proc. hat. Acad. Sei., 70, (2281 (1973). Fine structure mapping in the histidine operon. Bruce Ames. Method: This work is an extension of work already published to special cases for which the labor of mutant isolation and characterization is limiting. 1. Ames, N. B., Lee, F. D., and DUEStOn, W.-E., Proc. Nat. Acad. Sci. U. 5., 70, 782 (1973). 2. Ames, N. B., Durston, W. B., Yamasaki, E., and Lee, F. D., Proc. Nat. Acad. Sic., 70, 2281 (1975). Metal ion mutagenesis and plasmid curing in Salmonclle tychimurium. Peter Flessel, Assistant Professor of Biology, University or San Francisco, Sean Francisco, California ? Method: "I have been looking at the interactions of metals with bacteria using tuo assay systems. First, I have been studying metal ion mutagenesis and second, plasmid curing by metal salts. The decision to focus on metals was based on the ‘near presence of a colleague in the chemistry department who had been studying metal carcinogenesis for fifteen years anl was eager for some company. "The work to date has been basically an extension of Bruce Ames' scheme applied to metals. So far I have shown that MnCl and NiCls are mutagens in S. tymphi- murium. Our search for new metal mutagens is contiauing and I suspect we will find others in the next tev months. The mechanism of metal mutarenesis has not been thoroughly explored. It is not known, for example, whether metal ion pene-~ tration of the cell membrane is a prerequisite to mutagenesis. To find out, I would like to select for mutants which are temperature sensitive for resistance to metals. The assumption is that resistance would be a reflection of the fail- ure to take up the metal.. I would select for growth at 42 (permease denatured) and no growth at 37 (permease functional) in the presence of the metal. Having obtained such mutants I would test them for susceptibility to metal mutagenesis at both temperatures. I realize my proposal is perfectly straightforward. If I carry it out. with the time and resources at my disposal, it is at least a year's work. ith the "“dumbwaiter" I think I could have the first mutants in several weeks. ft. . tt ae t + - e = < . , - 7 _ Pd + C - . 4 Woes en virulence in Calmonolle and isolation of mutants suitable for «a 34 . enn eee eee coe ave vVacoun Bruce Stocker, M.D., Professor and Acting Chairman, Department of Medica wm ey : 2 ie ~ sytes Donald A. Glaser _._erivileged Communication oo. i? — Microbiology, School of Medicine, Stanford University, Stanrord, California. For experiments on the mapping of genes affecting the virulence of Salmonclla. species, it is expedient to obtain genctically marked sublines in particular strains. For example, in lines of S. typhimurium which differ-from the available genetically marked stocks of strain LT2 by their high virulence for the mouse, - on intraperitoncal inoculation. It has been the experience of several. workers that: auxotrophic mutants obtained by mutagen treatment of virulent strains of | Salmonella commonly nave unvanted additional mutations causing reduced virulence, by unknova mechanisms. Therefore, in theory, the best method of procedure would be to introduce choren negative alleles, determining nutritionel requirements. or inability vo ferment particular substrates, by transduction. To do this by {ordinarily available methods, even with the aid of penicillin enrichment, is hardly practicable, because of the amount of labor required to detect the rare transductants, which cannot be selected for. Dr. Glaser's apparatus should make possible the detection and isolation of the desired transductants by an automated st procedure. temperatures. a . 14) Isolate mutants of Bacillus subtilis resistant to certain phage and to drugs a like p. hydroxyphenylezouracil for studies on DNA synthesis. . A. T. Gonesan, Professor of Genetics, Stanford University, School of Medicine, : CO Pee t) BM at Seantord, California. : Our research project involves the study of the mechanism of DNA replication and ‘ ty 15) - oe ryt me S06 ne i we cyt ca ne ce ee nee eee oa eee fener ee meee em ot Privileged Compynication Coaqynwiien set oe 22 - . Donald A. Glaser.......---. Lo eee eee its genetic control in Eacillus subtilis, a transformable bacteria. We have isolated several temperature scncitive mutants that are defective in DNA synthesis The thermoscnsitive protein has been studied in a few cases. There are about 9 groups of genes that control DNA synthesis. There may be even more. We isolate ‘these mutants routinely by conventional, slow and laborious procedures. The automated petri dish machine would be ideal for the above project. We are speci- fically interested in both low and high temperature sensitive mutants, and mutants that are resistant to drunps like p. hydroxyphenylazouracil. This drug specifi-~ cally inhibits DiA polymerase III in Bacillus subtilis. Polymerase JII is directl involved in DNA synthesis. Resistant mutants vould help to locate the position ~ of the gene for the enzyme. The system is also adaptable to test phage mutants which are currently studied. The instrument is a very valuable and unique tool for our projects. We would very much like to collaborate with Dr. Glaser in obtaining several important mutants and adapting the machine for other related projects in cell biology. Sercening for possibl @ mutagens among environmental chemicals by mutations affecting sporulation in Bacillus subtilis. Lawrence E. Sacks, Research Microbiologiss and James T. MacGregor, Research Pharmecologist United States Department of Agriculture, Agricultural Research Service, Western Regional Laboratory, Berkeley, California. ‘ , Thousands of chemicals, whose biological effects are little understood, have been disseminated into our environment and the food we eat by modern technological. society. Most frightening of these chemicals are the mutagens, with their poten- tial for teratogenic effects, cancer, and unknown long-termn effects of alteration of germ-celle. : In screening for possible mut tagenic chewieals, microorgenism systems offer the ; advantages of speed, simplicity, and economy over animal systems. A disadvantage of microbial systems now in use is that they test only for mutations occurring in one or a few genes. A bacterial system sensitive to mutations on many. genes, scattered throughout the chromosome, would seem to offer important advantages over currently used systems (1). We believe such a system is that governing sporu- lation in the genus Bacillus. Sporulation is a very complex process requiring the participation of a minimun of 28 operons for the sporulation process alone(2). Other systems (e.g. TCA cycle) are required for successful sporulation. Eight ~ hundred genes have been estimated to be required for successful sporulation (5). Selection of asporogenic mutants is simplified by their characteristic white color easily distinguished from the wild-type brown colonies, colored by formation of a pigment late in the sporulation of B. subtilis,Marburg strain. Using a highly transformable strain of this organism, and a wide variety of mutagenic agents, many sporulation genes have been mapped (4) in programs designed to unravel the genetic control of sporulation. We propose only to invert this procedure, and to use sporulation mutants to identify new mutagenic agents. . Dr. Glaser's instrunent, capable of identifying single mutants in huge populations. will be of great value in identifying mutagenic activity at very low concentra- tion levels. This combined use of a bacterial system involving over a hundred genes with scanning by an instrunent cavable of identifyinz mutation rates below ' 1077 chords 3 “s4 QV crouse mrvbte tees dsr one meotssive rvos fax tdent aryt nS we wots g MD ae ee SD ey = muturenie choideals. PAs she ULaotl Privileged Cormunication =! We summarize below some advantases of the proposed. system: : t ~e , ‘Ll. It is based on forvard mutation, the mest general type of detection eyateri. - Any type of mutation which inactiviates or substantially alters a gene essen- i tial for sporulation will be detected. 2. A large number of genes are involved in sporulation (2, 3). Some mutagens ae are specific for particular regions of the DiA.- The more genes surveyed, the . .- less chance of excluding mutagenic “hot- “spots” . N 3. Sporulation mutents are often characterized by a block at 2 ‘particular stage in their morphological develomment. The frequency of occurrence of particular stages of arrest vill permit an assessment of the randomness (or specificity) of each mutagen. . 4, The Be. su subtilis system is well-suited to genetic studies. Highly transrorna- ble strains exist, and many genes have alreedy been mapped (4). Dr. Gleser's . scanning system, hovever, is not limited vo the pismented B. subtilis colo- nies. Other well-studied species (e.g. B. meraterium, B. cereus) may also be Lo employed. fe SS References ; ‘ 2 \ . tt i 1. Hollaender, A. (Hd.) (1971). Chemical Mutazens. Princinles and Methods for i i Their Detection. Plenum Fress g, iN Y. Vol. I, TL, III (1973). i 6% ! : . 4 Le ; wa I 2. Pigeot, P. J. (1975). "Yemnin ng of asporozenous mutations of Bacillus a j suotilic: A minimum estimate of the number of, sporulation operons”, " J. : ae Bacteriol. 114:12h1-53. Pe , ue 3. Balassa, G. (1971). "The genetic control of spore formation in Bacilli", ' Current Topics in Microbiolory and TmunoLocy 56:100-52 } : 1 4. Young, F. E. and Wilson, G. A. (19/2). "Genetics of Bacillus subtilis and other gram-positive sporulating Bacilli." in Spores V (H. 0. Helvorsen, R. Hanson, L. L. Campbell, Bis.) pp. 77-106. 3 me 16) Proline degradation mutants in yeast. ee John k. Roth, Associate Professor of Molecular Biology, University of California, . Berkeley. . We've started looking at proline degradation in yeast i ‘ny: . - Here the available mutant 4 : . —_— : men | ——.earichuent technicues, generally wor: poorly. Most pecple seek yeast mutants in a. | fairly “brutc-force" sort | of vay. A large set of proline-non-utilizing mutants would be very useful to us. This hunt would need to follow the Salmonella hunt and probably should follow preliminary work (in progress) on the few available ! mutants. In this way the most advantageous conditions can be determined. , : - i . . ; i :17) Saturation mapping of one yeast chromosome. . , ~ i John R. Roth Yeast has roughly 4-5 times as much DHA as bact eria- Roughly one hundred genes ' have been located. The snecine of these sence are wide enough to make it diffi- ia oe . ; . ne _. cul Soueh nove. Ag Senon (ey cia oo VoUY aubecaturetiion cc) WSR Pate Gh UR ON eh Wee OTP ED wal ty wVOULL give new moviora tor mapsing anti eo minire:: ertimeste of “tne mene density. te ae ce cp apnea ee OS EN ee ne faeee oe etme armament #05 20) A hunt for temperature-sensitive mutants should yield mutants carrying lesions in & ; ' Qcveloprent of genetic mass Donald A. Gloser Pantin tye mae ~ 3h - : . c. vie aes wo Tree Privileged Communication .oo i _=_™» _ we cessed cess Strains will be obtaincd through Robert Mortimer which are monosomic for a small chromosom?. Other strains will be: obtained which are monosomic for a large but ‘poorly marked chromosome. These strains are diploid for all but one chrouosome. the chromosome for which these strains are haploid. These can then be analysed. Determining the number of genes involves is fairly easy because of the simplicity of yeast complementation tests. I'd like to try this in a year or so after I've gotten back from sabbatical leave. (I'L1 be doing yeast genetics during that, tine.) . . Genetic mapping in Saccharomyces cerevisiae Robert K. Mortimer, Proiessor and Cnairman of Medical Physics Department, Univer- - sity of California, Berkeley. — oe re The availability of detailed genetic maps is an important component in determining the suitability of an organism for genetic and molecular studies. For a number of years, we have carried on a program of cenctic ma os in the yeast Saccharo- myces cerevisiae as an adjunct to our other studies. ese mapping studies have | resulted in a genetic map which establishes the lo cation of more than 150 genes on 17 chromosomes. However, because of the large number of chromosomes and the hign 7 frequency of genetic recombination in yeast, very few heavily mapped regions are | availavle. Such regions are important for studies ve wish to carry out on gene conversion and its relation to mechanisms of genetic recombination. We believe | the instrument developed by Professor Glaser could help to speec up the further in this crganisn. The avproach ve propose to use is based on the random snore te ecanigue deserived in our recent mapping paper (Morti- mer and Hawthorne, Genctics Te 2 55-54 (1973). A cerics of strains ¢ that each carry, one of a set of cchre sunnressors in combination wi ha 2 suppressible canavanine resistance gene and an additional selection of nutritional genes vill be crossed to a large series of tenperature sensitive lethals The resultant crosses will be sporulated, and the asci will then be treated with glusulase followed by sonicea- tion. The sonicated suspension vill be inoculated onto complete medium containing canavanine. Only spores lacking the suppressor and carrying the resistance gene will grow. These can then be transferred by replica plating to a series of "arop. out" plates to score the nutritional genes and toa W360" environment to score the conditional genes. The patterns of growth: nongrowth on these various | media can then be recordcd automatically by the scanner and the resultant data~* } analyzed for linkage by a suitable computer program. In this procedure it will te necessary to inoculate at a concentration that reduces to a low level the proba- bility of clones developing fron more than a single spore. The instrument should greatly facilitate random spore analysis both by permitting larger samples to be analyzed and by automatically recording and analyzing the results. ae ct t Gene conversion and recombination in unselected mitotic yeast cells. | eymour Fogel, Professor and Chairman of Genetics Department, University of California, Berkeley. Post-niclotic segregation and heteroduplex. DNA. . Seymour Fogel. f Tio rather specific pronosnls for use of the atometcd mierobioloyi.cal. equiprent are Peocented. Ghose aS tabhwes health rele: Ts, Che PoOaGe Se caepren sito: el LO: dn unseloeted Gerba. ecaregation in yeast conld provide a model system, a senvia Wy: adinsset o 47 4 ne Lat ea nas wea Tes radar wat yen wenn - we ae “3 te Feg yt tae ae ‘Our currcnt understand awa Tht . son pant ye : a ey prec ete cee POLE LVS CO t abe Cui > ' i sihpeueiy xy te ifs ~ 35 - - Donald. A. Glaser Privileged Commmicction SD strategies and overall rationale may be carried over to address such seemingly unrelatcd though central problems as chromosome nondisjunction,, or screening mutagens, carcinogens, fungicides and antibiotics for their genetic effects. I. Gene conversion and recombination i unselected mitotic yeast cells ing of intragenic recombination in cells committed to a mitotic cycle emerges fron data generated by selective methods. In effect, these: depend on appropriate signal devices that lead to the detection and recovery-of Only wild type or prototrophic recombinants. However, we have recently’ demon~ strated the occurrence of mitotic co-conversion in hybrids marked by three of four heterozygous sites in a single structural gene, and it must be emphasized that multisite conversions do not typically generate wild type recombinants. Thus, though co-conversions might represent the most frequent event class, they remain uncetected and unscored in conventional selective procedures. By. analogy to our studies on unselected couplete meiotic tetrads, we propose to analyze (in the same hybrids) an unselected vooulation of mitotic cells for all econversional events falling within a defincd genetic region. . Mitptic gene conversion in yeast occurs with an everase frequency of the order 10“" to 107°. Accordingly, collecting a sample of 10° or 10° unselected conver- . sional events involves sercening a total vepulation of 10” - 10% cells, ora sample beycnd the capability of routine microbiological methods. Automated ; microbiolocy equipment, however, augurs well for the successful completion of this and cimilar studies. , ¢ a) automated single cell innocula; b) replica- c) irradiating the replica prints (UV or X-ray); rieving sectored clones; e) finally, complete 5 ored clone by random spore or tetrad analysis of Our analytical setratezy reouire plating the derivative clones d) detecting, locating, and genetic diagnosis of each ¢ each sepment. Tr . ’ Bs e + & zr ec ‘ mee eee II. Post-meiotic sesrecsation and- heteroduplex DNA. Common to all molecular models seeking to account for genetic recombination are enzymatically mediated steps at eventuate in heteroduplex or hybrid DNA produc- tion. At the in vivo genetic level, the presence of unresolved heteroduplex DivA is detected by cost-meiotic segregation (FMS). PMS is comparatively frequent ~ among the total aberrant octads of Ascobolus or Sorderia. However, technical difficulties with these forms, including a paucity of genetic markers, preclude: total and critical analysis. With autorated microbiological procedures adapted to random spore or tetrad analysis based on diploid yeasts suitably marked with ‘(-lO0 heterozygous sites (i.e., loci and alleles of Imovwn meiotic conversion frequencies), we could readily assess the frequency, extent and distribution of heteroduplex DNA in the yeast genome on a statistically reliable base. Sectored ascosporzl clones, otheruice concordant for all segresating marzers will be considered as PMS events. a Lo . — Also, from the distribution of BitS events among spores produced by heteroallelic diploids of the type ++/12 or 1+/+2 (notation as before) where the mutant allele | pairs may be chosen from extensive fine structure maps to represent a range of i genetic distances, the resularities and bicie attributes of heteroduplex DNA . Oo nr Sy aad. a > Lat Oho Dia MTG er COS ue LpYerroou, tee awe 7 none weer ee eee ates Fee nee ete eee et eee tet ee ene ad ee 7 Ne eee: ees NT Se Ran tN es ame oe a ’ t : te a ee waetis Hepa eee 8 35a _ . - Donald A.. Glaser. ne Frank Ruddle, Professor of Biology and Human Genetics, Department of Biology, - Yale University, New Haven, Connecticut. FS I believe that your machine has particular possibilities with regard to the recovery of conditional temperature sensitive mutants in tissue culture populations. As we have previously discussed, it would seem possible to esta- blish colonies in the machine and then to shift to higher temperature and examine the colonies for retardation in the rate of increase of colony size. It would be possible to maintain the cultures at 34°C as a permissive condi- tion and then to increase the temperature to 38.5° for 3 hr. periods out of a total period of 24 hrs. and carry this regimen forward for a period of one or two weeks. It would seem to me that this would not kill the temperature sensitive mutants but.would result “in a decided difference in their colony size which could be easily monitored by your photographic equipment. The iso- lation and characterization of temperature sensitive mutants will, I believe, be one of the most important aspects of somatic cell genetic work in the next decade. It should be possible by this means to obtain mutants which affect the biosynthesis of cell membranes, nucleic acid, and protein. It is also possible to:pick up mutants which specifically affect the ability of mammalian cells to progress orderly through the cell cycle. All of these mutants can be analyzed by genetic complementation tests involving cell hybridization and Chromosome segregation. For this purpose it would be best to make use of Chinese hamster cells or mouse cells as the population in which the mutants are recovered, , It seemed to me that your machine could be adapted also for recovery of . mutants in differentiated cells. - Quite a number of tissue culture cell lines which express specific differentiated traits are now available. For example, we are growing hepatoma cell lines which produce albumin. ‘The albumin is secreted into the medium at high levels. -It would seem to be possible to maintain colonies and then test the individual colonies for albumin production perhaps using a fluorescent reagent. One can then examine a large number of colonies for cells which fail to produce albumin. This would represent -an excellent method for picking up non-producers. These cannot at the present time be enriched by selection techniques. One could also test for reversion to capacity to produce the differentiated product using the non-producing mutant as the base population. This kind of procedure could be adapted to cell © lines which produce hemoglobin, myocin, nerve specific protein, etc. ’ When your machine is sufficiently developed to make use of mammalian cell populations, I would very much like to be in touch with you with regard to these possibilities. If you are interested in pursuing these possibilities I'd be more than happy to come out to Berkeley and spend a month or so‘in this connection. By Privileged Communication =: — cette 20a) Mammalian somatic cell genetics. : Dee ee eee mee ee reper pam ee ae Corie oetian en 26. . Donald Ae Glaser. PDs pep - 7 O : ~ 7 Nw = Privileged Communication, — eee ey Ts solate and characterize a ‘larg a number or -steroid- and cyclic AuP-resistant ‘clones of mouse: lymphoma cells.: Gordon M. Tomkins, Professor of Biochemistry, Uni versity of California, San Francisco. : ; ' ae wane ne |. General Oojectives: For some years our laboratory has been studying biological ~~~ = regulatory mechanisms in cultured mammalian cells. We have concentrated primarily | on the action of the steroid hormones but more recently have become interested in the’ cyclic nucleotides as well. The bulk of our work heretofor has been a bio- : | chemical analysis of the molecular mechanisms of ecll-hormone interaction. Quite i recently, hovever, we have begun to explore genetic technicgues to pursue our ob- | : Jectives. For this purpose we have been using cultured mouse lympharn cells which: i are killed on prolonged. exposure to ‘either the adrenal Glucocorticoids or to cyclic AMP. Thic response occurs at vhysidological levéls of the effector’ molecules and i presumably reflects the well known irmunosupressive action of the glucocorticoids : and of agents which elicit cyclic nucleotide synthesis. In any event, we have beer. { able to sclect variant lymphoma eclls resistant to the killing actions of the steroids, cyclic AIP or both agents. Our results to date indicate that the transi- | tions from effector-sensitive to effector-resistant occur at random at a rate, i in.the case of the steroids, of 3 x 10-° per cell per generation and for cyclic i AMP, of approximately 1 x low? per cell per generation. Various mutagens increase the frequency of steroid resistant cells. Biochemical analysis of the phenotypes | of steroid- and cyclic AMP-resistance had indicated that in the former case, three types of variants can be isolated: those lacking the normal cytoplasmic steroid 1, _ binding activity; those where binding takes place, but in which the receptor- |) steroid complex is not translocated to the nucleus; ani finally those in which binding and translocation occur but céll death does not result. Preliminary investigations suggest that various phenotypes also give rise to ; cyclic AMP resistance. To date we have studied only cells in which the cyclic nucleotide binding protein and its associated kinase are deficient. | Specific Aims mo I.” To isolate a large number of steroid- and cyclic AMP-resistant clones of lymphoma cells. @. To determine the frequency of their occurrence and the effects of a variety of natural and artificial mutagens on the Uransi Grou from sensitivity to : resistance. . 2G 3. To determine the biochemical bases of cell killing. ‘ 4. To characterize the: PREnot types: in terms of various known steps in hormone action. - : 2 To carry out complementation analyses using cell hyoridization techniques to determine the number of piochemical steps involved in cell-hormone interaction. oo, . . 3 6. To determine whether the transitions result tron menetic or other types of i i - stochastic, heritable variations such, for example, as might occur during ‘ i . the differentietion process. ; : ; ' [. To investigate possible relationships between resistance to the steroids and | i to the cyclic nucleotides. -%. To apply similar methods to circuleting malignant cells in patients with { } * lymphoma or leukemia in an attempt to design more. rational therapies for : these discases. aoeniteonunes: Uhe projected sbecics bear on men sepects of cell IstoLory ant ee crm Reet eee 23) From a theoretical point of view, these experiments could provide novel approaches) Ceytinuntion ogee 3ST = - Donald A. Glaser... eee. Oe Febiilegsd comunicetion sD ene ae clinical medicine. The glucocorticoids are ma jor ‘therapeutic agents in ‘Leukemia’ ‘and in other malignancies. Their etfectiveness is limited only by the emergence of horuone-resistant cell populations. Our observations With cultured cells can’ ‘therefore serve as a useful rodel for studying how it might be averted. The finding that certain mutagens, in particular alkylating agents, enhance the con- version from steroid-sensitivity to steroid-resistance already indicates that ‘therepeutic regimes which employ alkylating agents together with steroids might be. redesigned to avoid the possibility that steroid-resistan t cells are produced in . the course of therapy. . v i ' These studies also suggest that new classes of agents, such as the cyclic nucleo-; tides or compounds which elicit their production, might be used in tumor chemo- therapy. The apparently lower frequency of resistance to cyclic nucleotides holds out the hope that these agents could be more effective therapeutically than ‘the steroids. . . So ‘ to investigations of drug and hormone action by combining geneties, with cell biology and biochemistry. It should, for example, be possible to isolate cyclic AMP-resistant: variants in which adenyl cyclase or various specific membrane recep- tors are deleted making it possible to study the interrelation between the elements in this important regulatory circuit. The same considerations hold true for the steroid hormones and studies on their mechanism of action. Steroic and cyclic AMP-resistance are the result of changes in structure of the recepuors. Since these molecules have been identified, and to some extent, puri- | fied, the generation of resistant mutants can be correlated vith altered molecules: Therefore a more complete genetic analysis can be carried out than if the selec-~ tive 1arker (e.g. drug resistence) were not correlated with a known protein. Linkege analysis in marmals by somatic cell genetics. Theodore T. Puck, Director, Institute for Cancer Recearch; Professor of Biophysics: and Genetics, Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center, Denver, Colorado. Preliminary discussion of this project has indicated the great labor of isolating mutants and establishing linkage. Feasibility studies need to be carried out . before real research plans can be made. Because the genetic exchange system is ! so inconvenient compared with E. coli, the automation may be even more. valuable for animal cells than for bacteria. Sensitive detection of muta agenesis by changes in colony morphology-extension to. additional bacterial and eukeryotic cells. : D. A. Glaser. : i Method: Since colony morphology is a highly polygenic characteristic, it should be a very sensitive detector of mutagenesis. Extremely uniform reproducible culture conditions are peauines to guarantee reproducible colony morpholozy even~ in the absence of mutations. For measuring gross mutagenic effects down to very low "exposures", we plan to explore the limits of colony reproducibility for a variety of organisms. ; ; ‘ succesrtul, colony morpholosy chantes provide a mechod of copie ePfucte on a wide variescy of clornable cells, even if Litile BEE . 38 _ Donald A. Glaser. . Privileged Communication >. ple sce a neers we tar mmeen caren coe thin sega eiennees Satie amano Hosa See ne ee ee ; or no genctic information is availeble. Screening of chemical and physical i mutagens is an “obvious application. . ; i : ' wo ee eee, 24) Transformation and mutation of Mamraiian Cells in vitro by low dosés of matagens and ionizing radiation. D. A. Glaser . s Oe ee rete mennntennter eon ang-E. J. Hall, Neture 25, 450-455 (1975). Exbryos of golden hamsters were ¥y minced and separated into individual cells groving on agar. The cells were irradiated with 1 to 600 rads of X-rays, incubated, stained, and the colonies forned 1 (probably about 2 uu in size) examined for forms made by transformed cells . , 5 j bo , “Transformation of Mermalian Cells in vitro by Low Doses of X-rays", C. Borek i ' t i j AO + eee meee Table of Effect of Transformed Cells ‘Dose — oe Clones Examined Cells Transformed 0 - %6,000 Oo | PS Po = 7,900 3 | , 10 , 10, 200 4 i - 25 oo 5,500 8 | Clearly large numbers of clones were examined for the infrequent event. The ability to use larger nuzbers of cells and examine the clones formed from them would make the numbers found more precise and allow better description of the dose response is curve at low doses 25) Behavioral Mutants of Motile Organisms —— D. A. Glaser ; ~ . . .- at mM. In the original provosal for construction of the DW and scanner system, we de-. seribed possible behavioral studies of motile organicms of standard or "instinc- tive" behavior as vell as adaptive or "learned" behavior. ‘The following is-quoted as an example of the type of study we would like to pursue sometime during the next few years. i ke ee ee ne ee eee oe, "Chemotaxis by the Nematode Caenorhabditis elerans: Tdentificat jon of Attrac- tants and Analysis of the Response oy Use of hivcants", S. Ward, PNAS 70, 817- --, , 821 (1973). Known behavior mutants of this nematode 1 were put onto gradients of an: attractant on agar plates covered with egarose beads or sephadex beads. ‘The i patterns resulting differed between the wild type and the mutants Some studies were done to understand the chemotaxis. The hunt for more mutants was. proposed. Clearly, in hunting for mutants, the more worms to be exemined the better. ‘The worms are small enough to be inoculated in 0.05 ml of licuid from an Eppendorf ; pipette. The patterns are formed quickly and photograph well. Analysis of the path can be done by computer in the same way Berg follows the three-dimensional path of E. coli. , . og we ee 26) Further Automation Instrumentation Development. 4 Although the Dumbwaiter and all of its ancillary equipment is expected to be in full operetion when this program-project would begin in June 1975, a number ‘ of specialized accessory instruments wae probably be needed as the biological : provram develongs. A sample of cuch Instrumentation projects that we envision . . ad. ot Pa rs oa . atu hie time Sollovs:. eee nee vee me epee Peete ee Rm eee jp mm square on the agar insteed of the present l0O-rm square. There is a trade-off between the time and cost of photography end the size of agar area covered. Optimizing the trade-off will require different magnif fications for different experiments. he Call Mantpuletion Deviets--Gur orarcent niens ere to use colony mielkors SIG, dint mileneing daevieot su eeclls that have sin ilar phys sical properties. We can well imagine uhge onher elip: vane . ecobites oo aa yee Sat Sos tine, py Fyn} ripen ane aca: vlabkion ayant LO ae Late} nF ee ty Nae tO ead stated ke y date oa ae = Tyce ey . wet ey yy sed onl oa sane re ctres Wabhe Jor colonies of BE. ecti end avaer special call menipwintion doviees wilh be reaubred trom time to ture. Donald A. Glaser Com tinstiow pose 0 LO « Meee UT nt test semis wep emee- -.. Privileged Communication... > . D. Significance . . . m8, , a ee In the discussions.above of the particular ‘biological research projects, the signi- ficance of each one was pointed out. In general, these applications of modern auto- mation technology coupled with corputer-directed pattern recognition and analysis of data offer a poverful new tool for accelerating research in a wide variety of fields of molecular biology and cell biology. They reduce enormously the labor, time, and =: materials required to isolate rate mutants critical at a number of stages in research ; as well as to measure with high accuracy frequencies of genetic and mutational events : which must be known for the genetic dis section of important biological processes. . eee In addition tO the great gains expected in the speed of research in fundanental _, biology, the same large-scale automation techniques offer great promise for a variety | ; Of bio-assay appli¢ations, including the screening of environmental chemicals ror 0 : their potential mutational and caxz rcinogenic cffects; the testing of proposed anti- ; biotics, antineoplastic agents, and cell regulatory substances.” In additional to the : i possibility of large scale testing of chemical agents, it seems possible to make highly accurate ‘measurements of the effects of ionizing, as well as non-ionizing, radiation on a variety of clonable cells. The information resulting is important to studies in fundamcntal biology as well as the difficult problem of setting safe - I “standards for allowable exposure to ionizing radiation among the gerieral population and among workers in industries involving the presence of radioactive substances. : ; With these large scale methods, it may be possible to extend the dose-effect relation- ship gown to very low exposures and- so to discover in an over-all sense whether there | A -, is a threshhold or minimum dose below which repair mechenisis prevent any detectable i ss of} genetic damage at the single-cell level.: - , . Finally, the success of these applications of the cutting-edge of modern technology 7 serves as a demonstration which may stimulate similar applications in industrial ; a :, a8 well as medical and research sectors. We already know of several projects for ‘ ~ ' strain inprovement of antibiotic producing organisms that have been directly stimu- : lated by this work. Representatives of a very large number of pharmaceutical manu- facturing firms, instrumentation manufacturers, and chemical compenies have visited our facilities. Suppliers of agar for.medical and research purposes have also visitcd our facilities and have discussed with us their problems in maintaining uniform repro- ducible quality in their product. Variability is a source of considerable difficulty © in both medical and research applications and we have agreed ina general way to measure batch to batch variations by its effect on colony morphology and growth rates | in an effort to help then anipreve the quality of their product. oo, ftom eee a eerie tere en eee ete The five-year period of this proposed program-project should be ample time to carry through successfully a muiber of the projects we are proposing as well.as to test the feasibility of a number of others and evaluate the usefulness of this kind or ; technology to biomedical science and industry. ee ee oe E. Tacilitics Available oO - : Virus Laboratory = Molecular Biology Department. ee i Many of the biological experiments described here will be developed, at least | i to the pilot stage, in the Molecular Biology Department and Virus Laboratory as has : been done in the past. All of the usual common research facilitics of these labora- ' tories will be available as necessary. In addition, a small, well-equipped machine Shep is av our cisvocal. . se 74 2 eet Me waa s ag an ‘ | | } 3 i ar “e avne eae, me nd oo jot a to | ’ i and vho have oP arene Games Yo neha 2 cme oe i F.- 3 wo Geert es wea i: : . ~" we - . Yr “Conti Eyes foe Privileged Communication - 41. " Tawrence Berkeley Laboratory oO: ‘From time to tine we may call upon special cor ewer te 7 mene “ Donala-1 A. Glaser —— aoe. wwe shops and: consult vith exerts. }-frev the Lavrence Berkeley Laboratory to help us with proziems which they may have already encountered in their High Fner _daboratory. We ‘Blectrical Engineering Departnent scanner are basement o7 and graduate students tae developzent and use of this SYTCGI. eering Department and the Engineering. Research Labore facilities for work of the type we.are undertakings, for those members of Anberested: in applying their special skills located in evecially remodelled *-Campus Computer Center - . Only nodest funds have been, budge ted y Poyzics and -} tion we can ofsen obtain electronic and o ther specialized L-prices and with immediate availability from the excellent s are very fortunate to be able to take advantaze -nical facilities and talent available at the Laurence Ber! keley laboratory. in’Mectrical ingineering have been taki Thus collaboration W rer use .of the Caapus Compu other orosrans. oom facil f the superb tech- AS Tne large-scale automatic equipment including ¢ the computer and flying-spot space provided for that purpose in the the Electrical Rngineering Building, Cory Hall.. Members of the faculty retories offers excellent s @s well as fas special @ unigve opportunity the faculty and greduate students in Electrical Ine gineering and knovledge to biomedical engineering ot . ' - F. ter Center ‘In addi- © supplies at very attractive - ilities cr the , per mene ing an effective role in: ith the Electrical Engin- eee since, until now, we have been able to carry out all the computations associated with our work on Our ovn computing system. magnetic tape and carried ont facilities for large-scale computational WOrk. oo. — Physics Department Laboratory space in the Physics Department When our own system is saturated, we may te eble to reorganize our programs so that some of the pure computation can bé put on at the Campus Computer Center which offers general a ~ is available for this vork it needed and the excellent resources of the Machine Shop and Glassblowing Shop can be used frem time to tine as necessary. Extra-fabrication Space . For fabrication cf much of the sheet me ‘the construction and mcintenance of the large-s granted the use of a corrugated etal building Hall convenient to all of our other crerations. this building was previously used for storing wining shaft. cal cals Loce af Collaboretive Arranzene nts ‘ all of the ecientific inves tigators v visited our racilities Since t relationship ou> oun labor: the precis oak nenbers of that will cevelop betucen atocy remain to be defined. a" PM ae te ce oe eee ae biter eee rn We ene ene Bete tee we we le Le wee sone. Oe Le “. aya ae jaye oe. pe mes noe eee mee , "% and welding work required for 2 automated ecuimsent, we have been ated in the parking lov ‘of Cory Coumonly called the "Ore House", ores obtained from a nearby practice ee ee “We have had extenzive conversation and in some cases correspondence. with vno have proposed projects using our eauipment the Cyclops has been running ' time, we have not encourz sed active york in our laboratory until very tle and igators and these scientii Fic inve in every case sc tentific RAS 8 Fe nee ee at at oe 10, Sete, ee Nee ne mee me eee ee eee ae ee : Bane a as y - : ee see ee only a short Aen er eeren wee: + + eee emmnein Foee e oma ers teh ne tee ee saben ae oe te ee ee net eee ee “investigators proposing projects listed here have independent support for carrying - out these projects in their own leboratories and we intend to provide use of our - "years. Donald A. Glaser oe ot *. : :.. 7 eestor eee gt tebe h - we ee tee oe : ca ao ~- e ~ . ‘ - a i facilities and necessary supplies without any formal arrangements or exchange of funds If scheduling of experiments and assigning of priorities becomes difficult, we will probably invite some of the scientific investigators to join us and constitute an Advisory Committee to help plan the work schedule. It is too early to. foresee accurately how all of these relationships will devélop so no formal administrative. structure for collaboration is being planned at this time. . In addition to the scientific investigators named above, Professor Hervert B. Baskin and Professor Martin Grahem of the Department of Electrical Engineering and Computer Science, University. of California, Berkeley have been very helpful in giving advice concerning computer hardware and software. They gencrously agreed to continue this relationship and perhaps play a more active role in this program in coming G. Principal Investigator Assurance. The undersigned agrees to accept responsibility for the scientific and technical conduct of the research project and for provision of required. | progress reports if a grant is -avarded as the result of this application 9 November 1973 ; mo, . mo TD) cialol Mba Date . Donaid A. Glaser , : . Principal Investigator che \ Vv Privileged Cormunication __.. — #§&» £4 voce eee ee we ees wee ee