Tc15;4wf. :;trr;itur h: - .-.....-^." . ..-.. -. --.------ . *.. - . ..-.- --.I----.---~-c--_--- il. F "._ I r e SECTION 1 DEPARTMENTOF HEALTH, EDUC;\TI0N, AND WELFARE PUBLIC HEALTH SEAVICF. LEAVE BLANK PROJECT Nl&iBER RESEARCH OBJECTIVES . w-w NAF~~EAND ADDRESS 0~ APPLICANT 0f3GArvizk7 13N University of C;;tlifornia Berlrci~:y,~~ Californ5.a 34920 NAME, SOCIAL SZCURITY NUMBER. OFFICIAL TITLE, AND DE?AWTIMENT OF ALL PR0FEssfONAL PERSONNEL ENGAGED ON PROJECT, BEGINFJING WITH PRINCfPAL lt..%ESTiGATOR Donald A* Glaser, S, PrGfC?3SOr Of physics Ronald Bsker, and Efolecular Biology, Virus Lab. John Bekovitzfl &soc. Dzvelopment c, Xngrg,., Virus Uboratory James Berk, Asst. Ikvcloi;ment Engrg., Virus Laboratory 02:!.lr.ent Zngrg., _, Virus Laboratory Principal Progra2mer, Virus Laboratorv John Couch, Ted Fujita, Robert Het:ryi I&f Hansen, ;i Develop. Engrg., Virus Iaborato,ry Sr. Dzvclopmcnt iQrg., Virus Laboratory , Principal Development Engrg.., Virus Laboratory (cant on n. -Scancer-Cc;r:;3r:i;er 22vcstigations of Xological Systems &j USE THIS SPACE TO ABSTRACT YOUR PROPCSED REsEAR`Cfi. OUTL.II\`E OBJECTIVES AND METHODS, UNDERSCORE Tfi~ f;~y v;0Rz; (NOT TO EXCEED 10) IN YOUR ABSTR4CT. . . iz Large scale genetic and physiological studies of bacterln, yeasts, and animal m- 2 cells gror;ln in tissue culture vi11 be carried out using recently constructed autcrzatcd ~~-z~i~er,t and com+xr-d$ectcd pattern recognition techniques. By automatic exami- 0 nation of up to 10 coIo&.e6.&'& &tch, rare mutants will be isolated and partially characterized,, mutagenic efrects . even at lo:.? dosx of chemical and physical agents will be measured anci genetic recombination frequencies measured accurately for mapping purpoL;cs. Nu'iants for detailed studies oZ DZA synthesis in E. coli and B. subtilis Km wi2.3. be isolated and partialiy chsractcrizcd. .lWants of E. coli, S, typhimurium ic and Saccharcmyces cerevicic/. dative patb:~:ays 0 a.; wil:L be ieolatcd L'or study of.biosynthetic and degra- WT end for analysis.of the mechanisms of genetic reccmbination. Genetic nap 01 E. . -. -2 coli, Saccharo:,iyces ccrevlsia-, and some mammalian cells will be enlarged. FeasiblEty studies 32 of automatic recognition of bacterial and fungal petho,gens 2n medical ani public health applications vi.11 be extended. ZE Irluta~enic effccks of food additives and other en*~iro,-i:,cnt8al` chemicals will be tested in several bacterlial., 4 yeast, and a&is1 cell systems. includj 1% ionizing t%xFnogcnic effec-ts of chemical and physical agents radiation wilbe seasurFd using animal cells. Interactions of hormoxs and other agents with tu:;or cells groxl in tissue culture will be cxmined to inve&igzke 0 Err-t-i-ne0plasti.c the biochemisal me-'*7 L,-,nism of the interactions and to test possible offec-ts of a variel;y of substances. ZE instant ,tu9or cells sensitive to s C~.v,C agents an$ resi.stant to otkrs Mill be isolated for further study. Screening. programs may be undertaken when Icasible for :Nikzg:cns, carcinogens, anti-neoplastic and effe i--t. 8 oi' lo?l doses of mut-,gen$ . Addftional in&ruxen-?&ion an2 ionizing radiation; xi11 be cons-kruzted as needed. 1.1. 2, . IJLB SC??- FROM THROUGH DETAILED BUDGET FOR FIRST 12-MOiVTH PERIOD I June 1, 1975 my 31, 1980 *: * -9, `..5. I DESCRIPTION (ttemizoi TIME OR AMOUNT REQUESTED (Omit cents) PERSONNEL EFfOfiT I XIHRS. SALARY FRINGE NAME TITLE OF POSITIOFJ BENEFITS TOTAL ,.I d&w*,.- ,PRINCIPAL INVESTIGATOR I See dctaj& or, eft&eii 3. 5t I I I I I` . I I 505.872 CONSULTANT COSTS I . I I Occaoional engineering consultarks for sDecia1 tcchnolo~ 1 1,500 EQUIPMENT I see details on attached D. 3b - I I 1828,200 , SUPPLIES . I .L I See details on attached p. 3c 204,6j2 DOMESTIC One major trip to Bst Coast for professional persons to TRAVEL attend a major coc?'er~ccc (Gordon, cm ( $2,000 ; attendance ij~ by professionals and grads at local conference (Arrowhead, Eiophysics); confer with colleagues and cquip.suppliers. j, 000 PATIENT COSTS l&e irtstructionsl .' ($1,000) * RLTERATIONS AND RENOVATIONS `ITHER EXPENSES iitamid 1 (See dr-tails on attached p. 3~) . I rOTAL DIRECT COST lEntor on &ye 1, /tern 51 INDIRECT" COST % S&WC j4.7. 96 TDC* 5-77 iSe0 lnsrructionsl , -I- 73 ' o fF THIS IS A SFECIAL RATE Ie.p off-sirai, SO INDICATE. NIH 398 (FORMERLY P~IS 398) PAGE 3 Raw. 1173 m UNDER NEGOTiATl~N WITH: .I 3a - Donald A. Glaser...-.- __ .:- . . .- . *Privilewd Conlmunication i.-. .-.... ..!? ___.__..._... . I - ~;IZR,"""'EL ---.. `T- - ..-----.. .i .----- ~ .-..-..-- .-.-... !..J _.__ _ ____ _ 1- Fringe Title of Fosition $%xe Salary ,Benefits Total 1 4 1. Donald"A. Glaser.1 iMechanical Engineers I Leif Hansen j John Bercoviti Larry Johnson I Larry"Henderson IShop and. Maintenance i Walter Debold I Lloyd Rwis . I. James Munger X ..' I xx (2)' - .'. icomputer Frogrammin~ i Fraser Bonnell' i x \ I x ' fcomputcr Operations I Robert Henry jInstruZZ~!~~tion 1 Ronald Baker James Berk j' 'Ted Fujita . Alex Fara .-~ I.-- Pat Donahoo. ;Biological Operations i John Couch ! Philip Spielrsan ( -Marilynn Brookm ! Carol Greiner f Eva Bennett I James Colby.. 1. xxx (3) i XxXx (4) Staff.Res.Assoc.11 100 -Staff.Res.Assoc.II 14,040 .-LOO -13,044. Staff.Res.Assoc.1 100 ,12,144 La.b.Asst.11 Iab.Asst.1 100 8,208 100 7,272 Post-Doctorals 100 35,5a Grad.Students 5076 - 9 mo 23,292 100% - 3 mo Lab!Helpers 100 12,624 - Prin.Fr0gr.V -100 24,612 Sen.ProSr. 100 16,656 Programmer 100 12,444 Sr.Dev.EngrS.V 100 24,oiz Computer Operator 100 19,906 Assoc.Dw.&Srg.V 100 20,748 Assoc.Dev.EnGrg.1 103 17,052 Asst.lkv.EnSrg.V '100 17,052 Asst.Dev.EnSrg.1 100 14,040 Leb.Asst.1. - '*IO0 - .7,560 . Aest.R'es.Biophysici& 100 15,290 . . IAdministration and Procurement ;. Madeline Moore ..- Adm.Asst.11 LOO- 10,248 ~ 1,230 Principal Invcs. 2'm0~. 8,652 PYin.Dcv.EnGrS 100 25,224 Asst. Ikv.RnSrS.11 100 'Assoc~.Dev.kgrg.III 14,748 100 18,792- Draftsman I 100 8,628 Prin.Lab.14ech.V 100 16,236 Pr3n.Lab.Eech.V. 100 16,236 Lab.Asst.11 v 100 8,196 kb.Asst.1 100 7,560 1,296 9,948 3,026 28,251) 1,770 2,255 16,518. 21,ob7 1,035 9,663, 1,948 1,948 983 907 1,514 . 18,184 18,184 9,179 8, W' 14,138 2,953 1,999 1,493 2,891 2,388 27,565 18,655' 13;937 26,893 22,294 2,489 2,046 2,04.6 1,685 907 , 2,294 1, a5 1,565. 1,457 985 872 5,335 2,796 23,237 w,o% 19 t 098 15,725 8,467 -v,5~4 -.15,725 14,609 13,601 '- 9,193 8,144 40,903 26,083 1 i -- . ._ _ _ _ . ,. -. , -Total-Salaries 450,084 55,788 il., 478 505,872 ~~,~::~.:se:ir:.? -.--:;T - 3b - Privileged Comrixnlcation -..- -*_-__- - --.... -- -.- .-.. -.."-.--------..----I-.--..-(-- IQUIPMZNT PDP 10-f: Systcn 'Unit Cost KI-10 . MF-10 RP-10 RP-03 IF-;0 TIGiOB TU-40 TD-3.0 TU-56 gc-1oa DC-1OB LF-l.OF *vB-1oc PD@-ll Pxocesfior 240,030 Idemory{ )641c wokds 80, ooo Disk Control 26,000 Disk Drives(3) so, 000 Wta Control for . . we.-. Disk and NagTape(2) 14,000 Mag TapeDrive Control 20,doo IQig.Tbpe Drive (2) 2 5,000 DEC Tape ControT 15,3w DEZ Tape Wives(Z) 4,700 DutaLiw Scanner 10,000 IHa Line Group, 8 lines I ' 5,500 Line printer, 1250 lpm " . 47,500 Gra.phic display 35,000 Controller for . 30,000 flying-spot scanner, (18 bits) Total 240,000 160, ooo 26, ooo 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 X.0-1) 736,700 o ,PD+ 310-I (Software pacl:ngc/p.a.) ' : `S,~OO : . Laser 4 watt tunable blue laser '. .' .i (Spectraphysics Eodel 164) '. . '_' .$,500 Flying&pot Scanner-`-speed up ,: PDP-11 - controller New faster yoke and A/C converters 30,000 10,000 PDP 10 to PDP 11 direct memory bus 37,000 Total Equipment 828,200 I Softvarc notebook updates ($03); sof6are updates .subscription ($1,100/~~); teletype pqer, printer paper, Calcomp plotter paper ($1,300); 14s~ tapes ($300); *'DEC tapes ($490). 'Petri dishes at $251 case (500 dishes per csse), 2 cases per week x '50 wks :: 5 lOO.cases x $25.. _ Agar at $1.3O/liter; 0.030 liters per dish; Id00 dishes per week"x 0.03 liters/dish x 50 weeks x $1.30 . 'v Agar for Cyclops trays-;20 trays/& x 1.5 liters/ tray x 50 weeks x $1.30 . Agar for Dumbwaiter-4 exper7tients/vk x 256 trays x 2.5 liters x 50 weeks x $1.30 liter .- .I- Miscellaneous drugs, chemicais, nutrients and glassware Film and D+evclopment $0.18,& for 35 mm fih Cyclops--20 trays/vk x 50 wks x 32 squares/tray . . x 6 photos/square x $0.18 ; @ photos/f& ? . Dumbwaiter--2 ?? ?????????? o ? ??? ???????? ? ?? squares/tray x 6 photos/square x $0.19 5 ` 8 photos/ft . ": . ,': ., .J10,.592 Miscellaneous small electronic, mechanical and . .optical parts for constructing laser selector-inoculator electronic controls, and new cell manipulation devices 25 j 030 3,400' 2,500 , 1,950 it 9% 49,920 5,000.' _' 4,320 TOTAL SUPPLIES OTXEi EXPENSES Computer maintenance ($2,9&8,/mo.--see'P, 3d) EE Kachi.ne Shop (2,030 hrs. at $ll/hr) LBL Machine Shop -* . . . , Machine shops (special jobs on and off campus} L?3L Supplies Phones Xerox Page charges5 15 .w at~$75fpwe : Publications - professional journals Mail $204,63i' 3y7; . 20: 000 10,250 9,600 . 2,700 1,800 1,125 500 100. TOTAL CTHER \ $103,451 XI-10 l?rocc SSOl KF-10 FP-10 RIP-03 m-10 TM-ioi3 TU-40 TD-10 .Tu+G DC-iOA DC-101) LP-1OF VB-1oc bair,tcnctnce pm? tid g>o, w Em3 . 00 79 .od ~10.00 134.00 43.00 316,.00' 20.00 : G8.00 19.00 B.00. 153.00 150.09 Total per xi0 2,348.OO DESCRII'TION .- -):+t* PERSONNE t COSTS CONSULTANT KISTS /kUir hi, travel, i,tc.) .EOUIPMENT `. SUPPLIES . PATI EN T COSTS . . . . ALTERATICNS AND RENOVATKINS TOTAL DIRECT CCkTS ,`646,G55 ADDITIO:.!AL YEAfiS SUPP~ 2ND YEAU 3XD YEAH -I--i?it( Y EAR -EAR 1 G-I-H YEAH 7fH YEAR t . * . TOTAL FCR ENTIRE PROPOSED PROJECT PER iOD Kntcr on`Page I, It& 4) -- I s 6,&4;g38 _ %%.?AFiKS: Justify ali costs for the fin-t yeer for which rhe peed may no: be obviwr. _-I.., For future yc?~rs, iusrify equipment co%, es wei! as eny tignific2nt increa;cs in any othf category. ff a recurring snnual increase in personnot cnsts is reQutzHcd, give percentage. (Use contincr~Okn p&g& if noc&d. i . - -+-co s$ of li&g .fQyired at: 10% 6y JUTle lcJ7.S. Etch ConsccAive year PS.~JIIX~ &t 105 increase for cost-of-liviaz increase and +sxSnPZ!atkon . . . . Xhployee bcncfite Pictxrqd at 125 fox+ non-acadmic; J.g.p,: _____,__ _, .,. .-.,s 'rd llor aa3derG.c alaries. . . . - SECTION It - PRiVlLECED ~~.~f~~~i~ATlO~ -5- - EIOGRAFIJiCAL SKETCH @JVU fh8 foJ\OWiflg ihwnstion fc~aNC;r~fr;s~oncJporscnne; Jistcd an ~Z~QS 3, &eg{nnJw 4th the $r{&p@] Jnvte;tjptcr. Use ccntinuxion ~s:ts snd follow t.90 s?rne gf?netz-J formor for each persona! NAME TITLE professor of Physics and' BIRTHOATE Ma, Day, Yr.) x-- -. Donal 3. Glascr Mofccuinr Bi.ol.o,yy 9;/21/26 PLACE OF 5tRTH fCir)r, State, CounrryJ PRESENT NkTfCrJALlTY (if r;an-U.S citizen, SEX. Cleveland, CMo, USA indimta kind of visa md expirarion date) USA a Male t] Female ._ EOUCATiGN (Eqiirl vrith bscceldureate training and ir;clude pnstdxtora/) , INSTITUTION AN0 LOCATION I DEGREE YEAR SCIENTIFIC CONFERREO FIELD Case Institute ofTechnology BS 1946 Mathematics and Physi California Institute of Technology PhD 1349 t1 tt Case Institute of Technology Sc.D. 1959 tt tt HONORS Henry Russel.Ar!srd, 1955; Charles Vernon Boys Prize, 1959; American Physical Society Prize, 1359; D. SC., Case Institute of Technology, 1959; Robe1 Prize (Physics), `1960; Ellio Creason Hodel (Franklin Institute) 1961; Alumni Distinguished Service Allard (CaLTech.) 1967; Gold I.!cdal Award (Case Institute of Technology) 1967. MAJOH RESEARCH INTEREST ROLE IN PROPOSED PROJECT Cell genetics and control nlec~nis~s Principal tivestigator RESEARCH SUPPORT i&Q instrvcti'ons) NIX Grant G!4 19439 d/1/79 - *4/31/74 Genetic Control of Cell $42,262 o NIH Grant GM 1324k 09 611173 - 5/sl/74. Physiology and Structure Scanner-computer investi- $405,698 gations of biological systems RESEARCH AbJJD#R PROFESSIONAL EXPERIENCE &art&g wi?hprcsentpositJos?,&t tr~;~~~nd~&pedeffcff reJevent ZOWBB ofprojoc& ListaJJ Of most mpresentativs pu.%icetions, Do not exceed 3 ~xgcs for tech individual.) Visiting Professor of Biology mT 1961-62 Miller Professorship UC Berkeley 196-z -64 Consultant Brookhaven Rational Laooratories, Argonne Rational Labo?atory, and a variety of other laboratories and agencies on scientific and instr~entation problems in physics and biology* .I* 2. 3. 4. -- 5* . . 6. C. B. Ward, 14. W. Hane, and D. A. Glascr, "Synchronous re-initiation of chromo- some replication in E. 0970 1 o coli B/r after nalidixic acid trektment," PHAS 66, 365 C. B. 1&rd and D. A. Glaser, B/r," PEAS a, 255 (1970). "Control of initiation of DHA synthesis in E. eoli C. B. 1qo.rd and D. A. Glaser, "Correlation betlleen rate of cell growth and rate of @$A synthesis in Escherichia coli B/r," WAS 48, 1061 (197i). - D. A. Glascr and C. B. ihrd, morpholo,&', Frontiers "Computer identification of bacteria by colony of Ikkfxxrn Reco&.zion, Acad. Press, 1:. Y. (1972). J. Couch, J. Be?%, D. A. Glaser, J. fizymond, and T. W&r, "Automated reco:?nition of bacterial s-l;rains by analysis of colony morphology", i?roccedings of the 13th International Congress of Genetics, Berkeley, California, Awst 1973. J. Raymond, J. Couch, D. A. Glaser, and C. T. IJehr, "Automatic selection of condiLionall&t defcctivc mutants of ~ic.rcorgn.nic!:!s;," Prodeediqs' of the 13th (continued) .-..--" .-I-v---cI_ w----e- . a i C.:;-!iP*b .)!ip : ;t-:.!c .I -6. Donald.A.-.Glasez, Privileged Corrimunication -. . . -. _. .- . -. . . .._. . _ ..--. .--I_._.__.,___.. ---.- - 7. C. T. Wehr, L. Waskell and D. A. Glaser, '"Isolation.and characterization of cold-sensitive `D!JA mutants .of Escherichia cpli Klz", Proceedints of the 13th Itlternat~iOnal'cOngreSs Of hXriiiCs,-B&teley, California, A&t&& 1373; . .._ .- -- 8. R. 14. Burger and D. Ai Glaser, "' Effect of nalidixic acid on DNA replication Sy tolucne-treated Sscherichia coli", Proc. Nat. Acad. Sci. 70, 1955 (1973). c 9. D.*'L. Parker and D. A. GlaserY "Chromosomal sites of DNA-membrane attachment< in Escherichia coli", submitted to J. 1401. Biol. September 1973.. 10. D. L. 'Parker and D. A. Glaser, "Effect 'of grollth conditions in DNA-membrane'. attachment in Escherichia coli:' in preparation. . . . . . . . Il. A. H. Dougan and D. A. Glaser, "Rates of chain elongation of ribosomal RRA ' molecules in Escherichia coli", submitted to J. Idol. .Biol., 1973. 12. L. Naskell and D. A. Glaser, "The isolation and partial characterization of mutants of E. co15 with cold-sensitive synthesis of DRA", in preparation. 13. D;' A. Glaser, "Some effects of .ionizing radiation on the formation of bubbles in liquids", phys. Rev. 87, 665 (1952). \ 14. D. A. Glaser, "Bubble cha.mber tracks of penetrating Cosmic ray particles", I . whys. Rev. 31, 762 (1953). - . . . . . 15* D. A. `Glaser, "ProCress report on the develbpment`of bubble chambers", Cimento 2, suppl. 2, 361 (1954). "- Nuovo .- ' '. . ; .' 16. D. A. Glaser and D. C. Rahm,"' Characteristics 474 (1955). of bubble chambks", Phys. Rev. 97 2 . . - l'l. .- D. A. Glaser, "Tne Bubble Chamber", Scientific American 1955. 18: J. L. Brow, D. A. Glaser, and M. L. Perl, Rev. g, 5% (1957). "Liquid xenon bubble chamber", Phys. 19. D. A. Glaser, D. 'C. R&m, and C. 'Dodd, "Bubble counting for the determination ., of the velocities of charSed particles in bubble chambers", Phys. Rev..%, 6, .x653 (1956): . . . . 20. D. A. Glaser, Decays of strange particles, Kiev.Confer&ce, 1959. '. . . . 21. D. A. Glaser, et al., 'I' The .Rev. Letters. neutral branching ratios of I;;" particles", Phys. _I . . .- .' . f .I 22. D: A. Glaser -and L. 0. Roellig, Phys. Rev. ~-6~ 1001.(1959). "&S-tic. +p and p-p scatteri.ni at 1.53. ~sv/c."~ . . . . -, : . - 1 -._ . -w..-. . 23. D. A. Glaser et al, 51, (1959). "Direct proof of, 0: neutral decay", Phys. Rev. Letters 5 , '. I . `1 PrivileCcd Communication *-.-- . ._^_, -. w .--. -.--s I. . . _*. d.-..--*.~.--------- I_-.-- i-~~~---.--..L.- and a computer-controlled flying-spot scanner", Ann. N., P. Acad. Sci. 139) 243 (1966). . * i ' . . . . i 25. D. A. Glaser, "Biological objectives and strategy for the design of a space i vehicle to be landed on Xars." Chap. 18, Biolo,~ and the Exploration`of Mars, I I .' Nat. Acad. Sci. Nat. Res. Council publication, 196o. - 26. D,A. Gla&er, J. WCatihy and 14. MnsPg, c'rap. 19, Ibid. (lg66).. "!L'ha auto~~ated"biological laboratory; i; I. . ' , .< . / 29. I i 30. i r 31. I f' i. Wolf, A. Ne:?!man, . *. and D. A. Glaser, "On the origin and direction of, replica- tion of the E. coli Kl2 chromosome" z J. Mol. Biol. g2, 61.1 (1968). EL L. Pato &nd. D. A. &Laser, ."Thc origin and direction of replication of the' chrorndsorne of Escherichia colTB/r. Proc. Nat. Acad. Sci.: 50, 1265 (1968). C. B. Wrd and D. Ai Glaser, "The origixi and direction of DRA synthesis in E. coli B/r. Pro:: Rat. Acad, Sci. 62, 681 (1969). L. . . C, B. -Ward and D. A. Glaser, "Evidence for multiple growing points on the genom of rapidly grorqing E. coli B/r. Proc. Nat. Acad. Sci. 43, 800. (1909). . I C. 3. Yard 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. 64, 905 (1969). . . - . .. _ _. 1 (Seeb,., e?inning of list for more recent publications.)' . ; ;,-- ~@BI.~ A. Glaser _ . _ ,._ __, I._ .-.-_ - -- . ..---- . . . e--e ?? ?? ?? ?? ?? ? ??????? o Privileged CommunicFltion __._.I-^___-...... _. _..._-__^.__ ". -_ . . -_- --.------- I . i I 8 I i Ronald Ba?:cr - Associate Development Engineer. b. Z/16/29 in Fulham, ~~~on,'En~~nd~ { u. 2.3. A-. Ruislip I&nor Sccon darn School, First Year Rational. Instrument ! I&kcr, LnSersolE! Ltd., England, 1951-54; Tool and Instrument Maker, G. E. . : Research Lab's ., England, I.354 -57; Mechanical Designer, .Physics Department, t U/of Kichigcn, 1957-53; I i' ! _ John Couch - Research AssociQte. b. 5/6/4x, Hartford, Arkansas.' NIT, SB Physics, i w ; 1963; S-kxdord, PhD Piophysics, 1970; 'Acting Instructor in Biophysics,, StanSord, -I *.-VT * 7" 4 r; i i- W.-R. Fair, J. L. Couch, N. 4ehner, Biochemical Xedicine 8 (3&-339), Purifi- , i. , `)c , :c i cation and Assay 02' the Frosl;atic Antibacter3.al Factor (P-Q); IIa1~:;ay&&;' H. ,rm +- + 1' and.Couch, J. L`I, "Thymineless death in Escherichia coli in-varioui assay . systems : viability d etermined in liquid medium", J. Bacteriof. 114, 228 (19'73); .f.k i J. L. Couch and P. C. Hanawlt, "DIVA repair replication in temperature-sensitive .h. p 52 ! .! . DRA synthesis deficient bacteria", Biochem. Biophys. Res. Commun. 29, 779 (1967); _. I i ; I 5. L. Couch and P. C. Hanawxlt, "Analysis of s-bromouracil distribzion in ;$.w I ;- i pwtially substituted dco:vribonucleic acids", Anal. Biochem. Icl, 51 (1971);' P. C. HanaT?alt, I). F.' Pettijohn, E. C. Pauling, C. F. Brunk, Dx1. Smith, 1. Y I 11) : L. C. Kamer, and J. L. Couch, "RepaQ replication of DN\ in vi_vo," Cold Spriki& . f .I I &.a ;'. .' Harbor Sympsiam on Quantitative Biology, Vol: XXXII (1968),87.. : :. . _ . . ` f"- `L! t's i z,Ted Fujita - Assistant D%elopmcnt Engineer: b. 9]19/&3,'Topaz; Utah. IJC Berkeley,. i !"' . BS, 1964; UC Berkeley, 1.33, 1965. p1-eject Engineer, Berkeley Scientific.Labs, . i 1965-63. . * . . . '. . . . . I . . . . :' I . . . . Robert' Henry - Senior Development Engineer. b; 6/8/3&i Xinficld, Kansas. ,. U. Kansas, BS, 1953; UC Berkeley, MI, 1965; Boeing Aircraft-Elcctrictil draftc- man 1357-58 (summers); TtILstcrn Electric CO., Engineer, 1959.60; RCA, Engineer, 1960-64. : . '. . N.d. % _f . Leif Hansen - Principal Dxclopment Engineer. 5/2$]27, Copenhagen, Denmark. . USA citizenship. Technical University of Denmark &!%E, 1954; Senior Design .. Engineer;. General.Dynamfc3- = Astronautics,' 1957-62; Senior Engineer, Lwrcnce Radiet$.on Laborqtor;.~. , I Bcrlrc!.cp> I.?&65. (!:.?dmic:?.l E?qi.%?"?r, RD.43 l@r-56). (continued):, 8. I:."!;:f*.!,?t "i' ;-`;,r' -9- ~~a;td A. Glaser-+. _.-_-_ . 1 ._ . . . Trivil~~ed.Co~lwlrication I. .-*. ---. . .._-. -- -- .._._ ---- ...-..---.------____IU . ..-.-___ ,___-..- -.-__-- -I&y Johnson - &.ociS&e &velopaent En&ncer, b. 2/&b7, Sfo~cc Fans, SOl&h I@!:ota. ST).SC~N.~~ Of Mines, BSME, 1959. ~,Sperry, ?rojcct @@.ne&,. 1959-65; Bpcinz, L)eeim Engineer, 1965-66; I;TiiC, Sr. I&sip X&&Ger, lg6&70; Thermidc::, Sr. Project Engineer. , Alex Para - Assistant Dwelopnent Xnginecr. be 2/?2/50, Buenos Aires,.Argentina. ',. Citizen of .4rgentinn. Chabot College; .&.A, 1968; UC Berkeley, BS, 1971; 'E;hgineer's Aid, UC Berkeley, 1969-70; Sr:Ehgincers Aid, UC Berkeley, lq70-'7$.' Sr. Dzvclopment Engineer, UC Berkclffy lgp-72. . * c ;;.;: :I a,,? : ; n - . :. . n IV : "J - - ga - ,Prl_vilcgc~..Co~~.unicgt-iQll. __ .__ _ ------..- llablC of Contents Justification of First 12-Month Period .Research Plan Sskcific A&, Methods of Procedure and Significance 24 Further Automation Instrumentation Development Signifikance Facilities Available Collaborative Arrangements Principal Xnvesti&ator Assurance 10 12. ., CT' - 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 equipTent. . In order to operate the computer facility around the clock, we will need to have two m-time computer 'operators, but no other major expense is. contemplated. For biolo- gical operations, a Senior Biologist with considerable experience in computer progrsm- 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. I 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 PDPlO-I System to replace our PDP-6 System. . Bd the time this proposed program begins in June 1375, we will have owned and operated-our present PDP-6 system for 10 years at an enormous saving in the cost of leasing the same equipment. Lease rates are usually computed to amortize the equipment in about 40 months and we will have operated the equipment for 120 months at:the same cost. Several years ago the PDP-6 computer became essentially obsolet e when it was replaced by the PDP-10, and then by the PDPlOdI system. Probably by June 1975 there will be a yet newer replacement of the PDPlO-I system; At the present time'(October 1373) there is only one operating PDP-6 computer left in the United States at the Band Corpora- tion who cLre 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 lbprovements fti 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-10 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 stillmaintaineb. 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 4 in analyzing photo- graphs from the Dumbwaiter and Cyclops. Since these instruments take photographs a'; ;;& l&e 0;: I- g?j: sccC::c. ;ltld OLW pCG:?i;$ rb,.i;c & e.na:Lyy,irg ptc.Lurps is a'r)Out 1 p:r 10 ccco;ldr; t0 1 pc? 20 SCcondc, blc hn.vc Z.il r:;:trc:r:ely unfnvol*abl~ l*atio of analysis time to production time for these photographs. With this additional -ll- Glaser, Donald A. Priviileged Co;;lt?uilication Justification of first 12-month period (continued) factor of 4 or more available in the PDP-10 system, the ability of the computer to analyce~dats xi.ll be nicely matched to the rate of production by the biological machines. For 'all these reasons, the switch to t'ne new system is extremely desir- able. ????? o This laser is needed to measure the light scattering ?? 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, :~je will increase the effective siie of the Cyclops and Duiibwaiter by a factor of 3 and be able to carry out critical sorting operations for experiments on animal cells. . _. Flying-Spot Scanner--spsed up. To further increase the speed at rW.ch photographs can be analyzed, WC propose to upgate the Flying Spot Scanner to cu?xent techno- logy by the substitution of the PDP-11 computer to serve as a controller for the scanner in place of the home-made circuit t&t does the job now. In addition ~!e uill 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-ll direct memory access-dxxp for bringing scanner information directly into the PDP-10 memory ljithout going through the slower I/O Bus. - Supplies--The cost of s:rpplies is'based on the asswnption that the Cyclops will con- tinue to operate. for small-scale experiments and for "second-pass" experimental 'material produced by lsrge DtZhhvaitcr expcrimente. It will operate with petri dishes or with glass trays at a modest level as described in the budget figures themselves. The budget for Dumbrxxiter supplies is based on the e::pectation th&t we KLl be able to carry out 2 batches per keel; for , cO weeks per year %:hich 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 n, c the Gordon Conference and the Cold Spring Harbor Conf crci3 cc, as ~11 as attendance by'professionals and graduate students at local conferences. Also conferences llith- co,lleagues and equipment suppliers. Other--Computer maintenance contracts are based on present cost estimates by the -1 manuiacturcr ~31~0 carries out the maintenance. Machine -shop time and other campus shops is 'required from tke to 't2.m :!hen our oxn single machinist is overxzorked or tjhcn special facilities and large machines are required for a particular job. The budget is base d on one man year of t:ork for this purpose. The Equipment Budget for subsequent years provides for new accessories bound to be required as the expcrixcntnl program expands, including for instance, a television- scanner system for on-line realtime analysis of growing colonies to eliminate the pho-koSraphy step and provide for the pass ibility of intervention in the experiment in real time and very rapid yead-out nzcessary for particular applications. For study of animal cells it zill probably be necessary to design a camera that photographs a smsll area of agar at a time through a loll-power microscope for studyine very small cloncs~ of animal cells, Other requirements of these kinds are bound to arise. Ve will justify this budget item on a year-to-year basis. Donaid A..Glaser _. . . , -~~~ivilcp3l Corrmiiunication . . _ . _ _ - . . . . - . SW.-- .__._._...I-.. _ ..__ -..-.---___ -___. Ykscarch Plan A. &xroiiuc-Len 1. ObJcctives.. Vhcn this t 0 a.?t ozk`e r!miy prosram-project began in July 1955, the.ovcrall goal wa 0 f the pI'OCZdLYft?S 0"; p&Xi disL technique on a large scale- using computer-directed mschincy;L end pz>tern rcco;;nition tcchniqties in a flexible &;y so that a wide variety of bio~;:edical-probicnls could be at-iiadked. 1973, after successful ope ration of several prototype's, Nov, ii3 November much of t?ie equipment is in operation 2nd aL1 Of the major equi~xxnt -Nill be in full operation at the end. of the curYen%' grant period in June 1975. t 'ik its &or': period 02 operation the machi&& has sueccssfully aided in the isola-. tion of co13 sensitive m;ltants of E. coli KL2 unable to synthesize DIZL1 at ZO'C. It has al20 ptn"OiTled hi&$lly ZLCCUY~LC? species of ba.ct&al pcltho@ens autwfitic recognition of gYOiliQ$ colonies of 10 iI:iipwtant in medical diagnosis as.a dcnonstration of its abilities in'msn&r health-rolaked applications. In thz next feu months we x&l begin nct7.f experiments in &nctic mapping, mutant isolation and,physiological chara'cT - - teriz.ation :jith 2. coli, Sa1t:oncll.a ty$zimuriu.:, Bacill:::s s&tilis; Sa&hxoi.yccs cerevisiae, and ani;:;aL cells Crwn In ';issw cul';urc. i,i&ny 02 *these projects will be done in coLLaboration:'12ith acier$ific investigators who have on-going projects in these areas. Wring the next five-year grant period beginnini in June 1975, we prop& to &ten8 these projects and add others involving the construction of genetic maps, the isola- tion of important mukants, and th,? eharactcrization of mutants and strains. Some of these projects will be chosen.to afd.in critical steps of the productive work of a number of independent scientific investigators alreedy working in these fields, and soxz KLl be important part- s 02 otzr 6::n biolo$cal j,rogrm:s. In additioil, szc propose to cxcmine tha feasibility 3f health related projects including screening of environxntal ~he;~?icals, including food additive- o for their mutagenic effects on bacteria, yzost and ani.nal cells, th2 potentkl. cc,rcinogecic and arki-neoplastic effects of various agents on animal cells, end'the cf32cts of very lo2 levels of ionizin~~radiation on various cCllC. ble and desirable, special fund- If larSe-scale sereoning projects appear feasi- 3 M.11 be sought to carry them out if necessary. Finally, a modest instr~wcntation program ::i.ll be conixinucd to add ne?l capabilities to the machinery as they becoxe neccsosexy. Scientists fro;:f Izany laboratories are expected to ta.kc advzntaC#e of this facility. 2. ??????????o? o Since tkis program includes a number of different biolo&.cal; projects, t&2 ;:~iol~Lcal background, rationale, aims, and n&hods xii1 be discussed project b:r project in slubsequent sections of this proposal. Vhat brings them toge- ther in this progrm is a similarity in the technics.~~nipuletions and the common requirement for large-scale expzrixents too tedioas, sloi,? and costly to carry cut by hand. In some cases aixantitative m=~~ ,-Gourements on C:ro:,ing colonies are extremely difficult without the automatic pattern recognition facility. Crcept for anal1 labor-saving devices, tcchniqucs for Cro?iinC colonies on solid media have chanSed little since they were icventod. E3ny projects in the contempor- ary biology of clonablc cells arc saverely limited by the difficulty of isolating particular mutants, characterizing them, and locating them on the Ccnetic`E?&p of the orCa.nisn. I3uxrous health-related pro,-rams including medical bacteriology; contami- nation monitoring; mass screening progrms for mutagens, o neoplastic agents; carcinogens, and anti- and industrial strain-improvement programs utilize similar tech- nfcue s . 1-t :rs hop::! "1 L.1a.l.d 'ihr;.z .i~~o-~~,7!~-pro~~cc~ -gill bc l.,'ei:l 23 j.n T!Qlqi; ill :,':vi$.C: );j!;tsl b:I o&r-\-;. uscPu1 in all these fit?l(?s a2 Privileged ComxMcation , _- --...a ---_. .---_---*.-_*..- -...--._-*-*-----I_. i I i ! t i t i i I ! i j i t I i ` 1 i t 3. Rationale of this automation. To carry.otlt, e$perimcnts requiring study of ! large n?unbcrs of colonies we are constructing a machine (the ."Diqfibf~aiter") in which i.256 4Ccm x 8'3~19 rgar-filled glass trays circulate in an.i'ncubator past stations where i various operations can be perf'orxed. An inoculation device deposits single cells {.carried in microdroplets in regular rows and columns for maximum uniform packing and.' 1 easy subsequent manipulati&. During incubation, tine-lapse photographs of the colo- I nies are made using up to 5 different colors of light. A flying-spot scanner.(similar !to a television c&r&) under control of a computer (PDF-6) .examines the photographs, finds all the colonies, and records their size, appearance, and growth rate. The\; computer then computes the frequency 09 various classes of colonies for measuring muta- tionratcs, map distances, recombination'frequencias, and other required biplogical results. In addition the computer can d+ect a colony "picker" to retrieve part of a colony for replica plating, suspension in liquid, restreaking, OF delivery to a test tube or small pctri"hish.for further manual work in the laboratory. Alternatively . the comP&er can direct the spraying of some or all colonies with nutrients or drugs on some predeter@ned schedule or according to.thc actual'pcrfo-rmance of each psrti- cular colony. Tnus.the computer can intervene in on-going experiments. Irradia"cvion, genetic crossing on the agar, and similar operations can also be performed as the trays move through themxbwaiter. Design of the DW (Dumbwaiter) and associated equip. m&t has been done to allot? a wide variety of accessdries to be added to carry out . special manipulations as they are required for p %icul& cxn=riments. If colonies, ale placed 1 mm apart, the DW can hold aLmost 10 8- colonies p& load of 256 trays. Several loads can be processed each day for many types of non-interfering experiments. . !:What does this kind of large-, ccale automation have to contribute to-biomedical science? ~Solution of.mnny biological problems depeinds'on theability to isolate a particular i 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 i conditions. Automation allows highly reproducible experitients to be performed with f:largc numbers of organisms so rare events can bc observed and more common events meas- ured with.high statistical accuracy. Computer-directed pattern recognition allows quantitative aspect s of growth to be e,xplored for regularities that would escape qualitative visual examination. .' _ None of this increased statistical and quantitative power reduces the n&&for thought: ful study of the biological system in advance of large -scale experiments and of careful analysis of the results. Xor is this kihd of automation likely to reduce the nurcber i or quality of people involved in a given research area. Rather the same ,people wilf be able to accomplish tasks impossible without the machinery and to do many.more con-' ventional e,xperiment s with much reduced tedium; In medical, public health, and industrial applications, large scale screening, con- tamination surveys and diagnostic assays, and other similar tasks can be done with the unbiased reliability of autolxtion and the economies of large-scale. It is expec- ted that these machines or adaptations of.them will be cost-effective and quality- effective for a variety of immediate health-related applications. . &. Comprehensive Pro,grcss Report. ' ' (a) Period covered by this report: June 1370 to h'ovember 1973,. ; (b) Sum;;sry. Equipment has been*built for inoculating up to 100 50 cm x 80 cm n'g~-flllnd~~3Sr;?~s. T:j.th sinzlc cells in regular ro:Js and columns of adjustable . fgnch~, il;cubp!iyij- _A ?A:: trays ;zn<.er tS<$ri;ly zon?.roilcd con:1itioas, ' . p~?o,co,?r:.7~~j;linl, them ~j2y~.C<.~cc?J.y, 2nd ar,$Qzigi; 4J;l.C i3~~3~O~~P~J?il!~ Tli<;;i? e sc:r.;:r?`?r-c~~pl~~;e3~ :;ys bi-. t. ""`7 1 .E%x:;~cn- ties of various colony types are recorded by the computer which can also direct the Dxwld A. Glazer automatic picking, replica'platin& and restreaking of colonies it is instructed to select. Nutrients, .drrss, viruses, end other apxtto can be delivered to whole trays or selected oolonies un.der computer control. &sign and construction of a fully-automated system able to carry 108 colonies on 256 trays is near completion. With the,. presently operative system :!c have isolated cold-setkitive mutants' of E. coli Xl2 unable to synthesize DXA at 20oC using l/5 as much azar and much less labor t&n _ a pa.rallel projcct using hand methods. Ene bacterial species isolated from human urine and a laboratory strain have been studied ??ith the automated system; Using neLly developed programs, the computer can correctly identify unknown colonies of these ten types Mth ac:uracies better than Ss$. A. h(c) &tailed Report 1) Biological Projects. Although the goals and budset of this program-project we& directed principally to:,,ard development of the autozxxtion system, several biomedical projects have been carried out to demonstrate the abilities of the system and to speed the work on biological projects in our laboratory supported by NIX as GM 194% (replacing C&I 12524). The NIH 'preferred funding the instrumentation and biology prg- grams separately so that they would be reviewed separately by appropriate panels. (a) Finding, Counting: and Sizing Colonies. Computer programs have been written for scanning photographs of LOO-mm~i_setri dishes preparzd by.hand for finding, - - counting, and, sizing the colonies c&rectl~~,in spite ozi" overlaps of colonies and xLde variation in colony sizes. The counting algorit!3l_r has an accuracy of better than 9$F' ,J on dishes containing up to about 4GC.colonies and-requires about 10 scconck per dish. It isathus greatiy superJcr 'io any csmmorcial colony counter, but is not used currently bkmse simpler and faster prog%&i5~~are effee- tive with. regular array-inoculated dishes and trays. (b) Isolation of Cold -Sensitive %.kan&' (by a method widely applicable .to mutant hunting). InvcztiCations of i3XA synthesis in E:. coli, its control, and its connection with cell divisioh, require isol ation and genetic mapping of conditionaXLy l&ha1 DXA rutants. Work on OLE laboratory and many or;hers has uncovered 7 or 8 cl asses of heat sensitive mutants normal at 30oC or 37'C but 'unable to synthesize DITA at about 41'C. These classes map at 7 or 8 distincti" sites, but probabQ- LZA synthesis is even more complex and additional-sites defining &ore struckrtml or con';rol'gnks remain to be 'discovered. We are searching for nw classes among cold-sensitive mutants unable to synthesize DI?A at ZOoC by taking time lapse photoCraghs ai" colonies Ci;ro>!n from mutagenized cul- tures and shifted betileen 20oC (restrictive temperature ):and 37'C (permissive . temperkture). Imposing conditions on colony diemeters and growth rates leads to efficient selection of cpld-sensitive mutants by the scanner in a ??ay that saves much labor and materials r;lhen compared Tjith a competitive hand experiment run in our laboratory. Cold sensitive mutants mappins at a known site (C class) have been found and 3 nw mu-cants may represent a new class not yet precisely mapped. Since the colony piclre r is not yet in operation, 'the scanner aids in locating mutant colonies by displaying a map of each dish on the display scope. HoldinS the dish a@.n& the screen, mutant colonies are picked wherever the co?~:l'sc?r ha,s draw &n X. l--c: ,::..,;i yr,?, :`.`f"" 15 " Dgta1.G.. A. Glagq Privileged Coz71unication .- .-.ml-.- ,.____ _.^ ,_-. ..__ I ._, _..- _-. ._a.-.--..-.. ---.-.--.--'----~... &.T . ..---I-- --e..- _.._..__.._I__ __._._ -.-_-. . -. .This method (.later-using ttie picker i~"eliminatc hand lgbor) can ue used for 'eny mutant selection baaed on colony size or o.ppearancc. W& have,tested it success- fully with ~EOLTI lsucine aE:ot,rDph% by gr?iaing mix cd prototyophic and auxotrophic culturcs'in &:iting lcucine and then spray.inC: the agar with additional leucine, taking phOtO,-kp W- hs during the incubation intervals. . ' (c) Automated ,` RecopnZtion of Bactericl Stratns 'by Analysis of Coloz~y Xornhol=t;r;lr. To test the ability of the systcn 'GO identii'y bacterial patho:gens for medical and .public\hcalth applications, we photographed F&hour colonies of nine spec5es ikolated from human urinary infections pLus one Eacillus subtilis strain.' Using methods of colony mornho1o.g ar?alysis dcocribedbelo~, tile system "learned" to ~reco@.ze the 10-test-spcsfes by k---p' iLa+nfng abo*ut 1090w colonies of each. tipon .scanrfng an kqlditional LOO0 colonies presented in mixtureo or in pure cultures, the~program mai:kk t:ro dccislons: 1) whether to attempt an identification (ans- wered "no" if the "colony" is nbt round, fs actually a piece of dirt, an impzrtcc- ,- tion in the asar, etc.),.end 2) t6 r&at species does the colony belong (5x3 1) is.` ans:>ered "yes"). Results wre as follows: _-. . ..' $ AttemptFd Aerobacter aerogenes 3acilJ..us subtilis Escherichia coli Herellca va@icola Klebciella pncumoniae Protcus morgwii Pseudomonas putida SakzoneZ3.a typhimuriu7x Serratia marcescens Staphylococcus cl,meus 83 ;;: - . . 87: 86 83 882 89- $ Corr& 100 : loo loo 100 98 100 100 100 _ 100 109 2) Technical Pro,rfress. Vhcn completed in January 1975, our automated system ttill prepnre minimal agar median in 4004iter batches, dispense it xlith a progxx~ablc variety of additives into 256 40~111 x8Ocm prestcrilized glass trays, and circulate. the trays inside a precision incubator past stations for inoculation, time-lapse photozrapkJ, colony picking and replica plating or rcstrea?dng, and treatment 175th chemicals, radiation, viruses, etc. 3-1 January 1973, the prototype test -version came into opcrztion and is noI+ carrying out almost all of the operations of the final systen semi-aaLoxai;icaL~~ on a reduced scale (about 100 trays rnaxizwa ea>ccity). "' Photographs are exix:Tlincd by a flying-spot scanner (similar in operation t0.a tele- kision caEera)'.connected to a meditx-sized computer. The computer finds aIlL the colonies, measures their diameter, cheracterizes t'nelir appearence (using up to about' 100 paranetcrs), and issues ccmmands for colony picking, nutrient spray, h.tant ,purification by colony re,b c"reaking ank? replica plating, according to ti protocol - written by a biologist. . . . By Juk 1975, when the presently proposed program is due to begin, the s?;'stem should be in full operation. Technical aspects of this ~tor k have been reported in t??o piiblisht?d papers: D. A. Glaser and 17. H. Ilattenburg, An automated syfstem;for the grot!th and analysis of large nwbers of bacterial colonies using an environmental chwber and a co~pl~ttcr-cont~~ollcd flying-spot scanner, Ann. N. Y. Acad. Sci. 3.39, 243 (1966); D. A. Glaser and C. 3. F?ard, Computer identification ofbacteria by colony norphol.ocr, PFontkrc of Pat'tcrn'Rccorn~ti_o!l. Academ5.c Pxzss; 113. Y. (15172), - --I ,-----L--L *-.,w--.. *.. .._.. -..-m 4. .._-- .- - ..-wC.-- . . .- . ---. .-*.u- .--- - .-...- -- . ..-.-._- l..,. _ .-__ 3.13rge number of or31 reports, and Prozrcos Reports to the T;JIc;EB. Wtniled -publications will be,*prcpared after the'full sy+ d&m is completely oper3tional.. a? h3ve seen no rc;o.rts of similar systems in Qierztion clsc:.4~erc. _. .* . (i) 00 jecti~.res -_ and General Descri$tion. &lar~ of the biological objectives of this pro@rar,~ rer~uirc the ability to examine about loo fairly well isolated nies (about one colony per squ3rc.centtiMxr). For other studies up to la8 eolo- colonies need to be ex&ned but they may be cro:jded into a ~naller~space (3bout lq0 colonieg pe; square centimeter). The mschine must therefore have a capacity of about 10 cm of solid Gro$h medium (agar, silica gel, or other madiwz).'i To provide the required area of ae;ar in the.sma.;lest possible volume, the machine' uses stacks of horizontal ag3r-covered trays epsced one inch apart. '!These trays are made of inexpensive w%nc?o>? glass vith mete1 frames 3nd can be rwhed and &erilized v"ry easily by reasonably standard techniques. They 31~0 provide a very uniform gro:?th surface of high optical`quolity so that good photograpl~s Of' groi!ing colonies can be made. Ordinai-J pl3stic or glcsb petri dishes made by hand in the laboratory in sr?all batches in the convention31 way can be laid on the tr3ye for incubation, photo,gaphy, and manipulcition in the machine. Al- ternatively, large-sco.l.c experiments can be carried out by pourin 3 sheet of : agar directly on the tr3y. A dcsi;;n has been chosen which cakes it possible to intermix these tuo modes SO th3t the petri dishes mzde by hand can be analyzed at the s3w t-ime as large-scale experiments prepared au~omatic3lly by the ma&ix The entire machine is foully automated to perform large-scale microbiological e::- periments in conjunction with a sophisticated data gathering and processing .' system. Bec3usc the stacks of trays 3rc moved up and do:?n by mechanical devices, we have called the machine "A &mb%+xiter", The design concept of the D&mbwaiter is very simple. Glass trays czrried in 3lu;ninum f'raws are steckcd directly on top of each other in trio stacks about 25' apart. Cross -ducts areprovided to tznafcr trays from the top ox one stack to the top of the other, and from the bottom of one stat!; to the"iottom of the other. The trays then circulate in a rectan&I3r path moving up through one siacl; across to the top of the other, down,throwh the second stx!:, and across from the bottom o? t'ne second stack back to the bottom of the first stack. !Fhis over-311 design p3.3n can be seen fn the attached figure. On t!e cross- ducts for IZOV~~~ the trays horizontally will be mounted camer3s for photogaph- ing the trays and spcciel 3cccssores for inoculating the esar vith organisms; administering drugs and nutrients,' r3di3tion, picking, irradiating with ultra-violet light or other 1 restrcaking and replica p$xtin~ colonies and other manipu- lat ions. !L'rs~ys are handled singly only in the cross-ducts. 21 cvcryrtther part 1 of the Dumburziter and auxiliary equipment, the trays will be handled in stzcks of 54. T;lC stacks 3nd transfer path s are enclosed in housings in which's sterile. ~ro~~th-environment is maintained. Mi?sing~ sterilization, and pouring of agar is carried out outside of the Dumb- TJ3ii; CT o Accessory stations 5~il.l also be ,movided for wshing the tr3;cs for x-use, for sterilizing them, and fbr special incubation, and cold-stor&e of . trays of colozies which do not need to be photoSr3phed very frequen-bly. Four moveable m323zines willbe provided for storing stacks of tr3ys and transporting them from the Dmln~3f.ter to and from various'auxiliary stations where these special operations M.11 be cslrried out. Tne separation of these necessary func- tions to 3 number of specialized stationa*.w Ls i'ound to be.the best wy to pro-Jlcie ?:2p:,,,, -r~~?-$:~.i~.l.e ord crn::3::j.-.2!: 03nm+;ieon `:?? -kltc; _c ..&-i,.. -. -7 r?.&?,rc `.cvq `ye :!.;]..z c,jrrp $,i-TT iJ.$J,?:;_lr, &--,t& 02 th.2 -folkwb-i~~ pnge:: . ,?:;*j, ~~`.,l-., -i,,.-,c?~~:-ri~`~):`Lcls I>.:.' ,k&;, Dr-r.:>*~",i.p-- a:ld i-:s .v a- ..-_- w -.I, 2 Lt-.-;7 i2.y. - -.- ; c; <, , 1.: ..T 1" .,_) `I . i.`-.L . . . . . d..". DmaZd A. Glaser (b) @crat:ionrl protot~~,~e (Cjwlo~)s): As.deaign.a&.testing of Dumbwaiter corn- ponents procee:is, WC often need to construct temporwf devices for testing dc- 'sign principles I . end mechanical ciwi.ccQ c that ?lill SC used in the Bmb~aitcr. At the same t5_rrR ,.-, ve T?ere anxious to begin carryin m out biological experiments before the kmbrraitcr comes into full operation. We haye, therefore, constructed 2 machine called "Cyclops' consisting of a DJr;b:zaiter camera mount&d on da x-y motion capable of handlin, 7 one or tt;!o Dkbwaiter trays in the same uay that ~lill .be..done in OQZ of the horizontal cross-ducts of the completed Du.mb%?aiter. Cyclops is' capable of photographing a.gar-laden glass.trays or trays carrying conventional plasticpetri dishes, of inoculating sterile agar with organisms to be gro;.n,. of spraying drugs, nutrients and other substances, .of picking and restreaking colonies and carrying out most of the mechanical and optfcal operatiotis of the Dmb%:aitcr. . It is not- able to incubate and circulate trays, however, end at the present time Requires the trays to be transported by hand. fiTearly all of the other ancillary facilities of the IWbkiter are being used routinely for eqcrf-i mcnts done on the C'yclops as ~~5l.l be&scribed below. (c) -Moveable maL~azines. Tae moveable magazines serve many purposes. Tneir main function is to transport and protect the &-tray stacks. Ezch stack rests on a dolly on rails on the bottom of this magazine. %hen the moveable magazine is engaged to a'fixed magazine for transferring a stack in or out of the Dumbwziter, the rails in t'ne moveable magazine mate uith cor?csponding rails in the fixed magazine. llie movcablc magazine and the fixed magazine both hzve doors facing each other. The space left .betueen t'ne doors after engagement is accomplished uill be sterilized by UV radiation; !i%c dpors Ml1 then be coupled together 'and simtiltan~eously lii'tcd up into an enclosqd UV irradiated container above the fixed magazine. The lifting of the doors i, * performed oy an air cylinder. The .staelr trans5"cr can no*;z be executed using a hand-driven transport screw located in the moveable magazine. . .Nhenever actual stack transfer is not taking place, the dolly is locked in a ~ fS::ed horizontal position and the stack is secured in vertical compression by hand-operated scrc;-!s in the ma+gazine top cover. This will prevent uwanted move- ment of trays in the stac!c during traMport and handling of the magazine. Tile vertical compression ~5.11 be especially important to keep all trays parallel to each o"ther during the agar-pouring and annealing process.. The agar+ouring will be done while the rvx~eable nagazine is reeting on levelling jacks in a combinatl.on sterilization, .pouring, and annealing oven. The stack dolly is '7 equipped with mercur, lr levels (permitting .180oC dry sterilization) to assure accurate lcvelling of the stack before the agar pouring. 2he agar;pouring prows. .enter the moveaule magezim through two rutozrticelly sealing. vertical slots in a side wall. The moveablc magazines have no thenaaf. insulation and they do not h&e angl.temperature control system. Tne moveable magazines M2.l be transported olltlan &r-cushioned transport pad of adJustable height. .All four of the portable'magazines for the final Dumbwaiter system have no%]. been completed and one test model dubbed "Oddball" has been made for e::periucnting with control of temperature and humidity in the portable magazines an&for use in: &?KlihdXiX%t2C operation t0g.$SX?r tlith the Cyclops, Oddloall' is in constant use in connection with Cyclops and is,perfoming well. ball-ccre:1 *xLth- attached nut.. !iBc ntik carries two- acar-pawing probes rlhich go thros$1 the slots in the naza, nc. -i land oscillate in and out th;~ou&.tha vbntilatlc hdles in the '.trap . Both the mixing plant and pourrng device can'be autohated ca.s~Lly using a PDP-83 C6mputer as the main control element. The ggar. plnnt is a rather elaborate sy~3em for nixing it'sterile into the DumbTMter.. 400 liters of agar in a batch and delivering It 5s compktc and has been used several times for pouring .-teat plake., q T!hich sh.o:red no contamination, @led perfectly, and showed SocJd optical clarity, ,It products- ~XJ liter b&h&s of neutrkl agar con- taktin~ a ninimuzz addition of salts. The a,nar will be dispensed throu&ahoPe in the roof of t`ne sterilizing oven into the tray-R.lled magazines, thrkxgh a manifold zthich xill allot,.thc addition of specific nutrients, carbon so~uxces and other additiws wdcr compxkr c,ontrol. In this key it will be possible to prqarc a fqll loed~of trays for the Dumbx&ter containi& a variety of diffc- rcnta~ars for.`thc sbultanzous pcr'formanzc of 'several cxpericents~at one tytie. It is not cconomical.to LEO the ggar plrznt on the reduced scale experiments beirg carried out a%,.the present time. .:. . (e) Steri_bi:x!.n,~ tire n. A large oven neckssary for sterilizing glass 'trays in . their uovcablc ?cgzzines for the use of the Dumbtziter has been completed and is routinely in use. It holds a temperature, of 175OC for eight hours for dry sterilization of stacks of @.ass trays held in the Wdball noveable magazine as it is used nolj aith the 0~~10~s system. . (f) Automatic cgar dispensing system, Under the control of the PDP-8E computer, the autornei;ic a;;ar-dispensing system can dispense enough agar to fill a glass i trny in 56 s~~co~rxz,)` without kplzchi& v.sin g only fouY electricallfr-controlled- _ valves. Also under control of the PDP-8E computer'is a mechanical system using a vertical ball screw stepping motor combipation that indexes apair of nozzles vertically from one pair of trays to thc.'nc:rt through tile s-teck to E.,~-;Jlcn in order. After all the trays have been filled M.th a&, the tcmperature'in the sterilizing oven is gradually lottered so that all trays have the same annealing cqericncc. !&is Is Sxqortant S, provide uniform and reproducible agar surfaces to all of the orgmisms in the subsequent .batch experiment. (g) Const ent tmymtwe roox. FoI.& rooms ha.va been constructed at one end of the l.?%%?~ory to stccox.~oda'~c moveable magazines from the Ikx~buaitcr t&t carry the equivalent of 5,001, petri dishes each. The rooms have been tested and are able to hold a tecgerature in Vie range of 0' to 50oC G.th sn accuracy of '-3 + O;l'C! or better. This arrangement permit s incubation of dishes at a.variety 5-f tenpzra%ures as ~211 as .cold-storqi n of.those dishes that must be held before the next step of processinS. The rooms are.in use for small-scale experiments. : involving the O~clop s system. and the Oddballma;gazinc, ,as pell as -for other .ezqeri;cents carried out by hand. Precision controls and recording appal*atus for the constant tempcraturc room al101.1 the experimenter to krxw the exact status of his Incubatin,g culture- at all times. (h) Photog'aphy. ColoniCs are KLlum~nated from beloz! by a very stable source of very parallel light provided by a high-pressure xenon arc lamp and,-+ 7" off- axis paraboloid nirror. 931~ camera is provided with an auta;&i.c focusing device which changes the focusin, v distance up and doFIn to compensate for chatiges in the thickness of alar, in the pl;?ccnont of dfshc, p and trays artd other sou1'ccs of ~~cc:!r??lr`c~.l er-nl' 'ihzt e0LJ.x ?-m`xz SlS.~$t Ckiin~2S in t;IlC c!-i&ancir-s fro2 the cmcxa A. "0 GIL ::~;c.r c~l.y:~,?./~-t. _ I :zp ,:~it;fpy,':?. ia l;f:n+; ijj ::'o~u~ y-i.i;ji 521; a,pf,:!?',*:+\?;; 3:: -J- O.(XJ5" j.n ::,n$-k: 3 i' `;;;:,nL.r_ fO&.;`L;..JC p::' :`-:~yj-~~i>!:. * *pc :,r.-Yc L`.. 1 Ld fJtc$y-Jnje~3:: ~.;.:~-.!.r~~.,-~~~-?.ilut"ie?-s - .., 1. ,. ,- wdez the control of an integrating 1.2&i:: qeter .dssfgncd to guar~p" --see reproducible .c;cpoeLLres 'from, one pickurc to the next. tcm as vcll.as~`&2all uricertaintieti -To carl:ect for slight' errors iin. the sys- in 3-k unT~orr;iit:: of %lre ori&xd. nliotograp?iid materials as veil as tl?e su5.seqwnt dcvelo~-in~ process, a "step ixdse" of variebl~ optical. density is p;?otographed in "uhc, corner 05 cac`n pIct& so ths,t subsequent measurements on the fiti of the image o:P thLs grcy ExJ~~ uill allov all the data to be reduced to standard e:~osux condi.tions. ters of iarious colors A color :?hcel carryitig fi.vz fil- Op3-a'iCS in thz illu.min&ing light bear? so that black ani T,lhite photo~sa~5s can be take n under computer control of any clhoice of f3-vc i` colors. Fhally, the ca.xra; has btilt into it a data board carrying an array of binary coded li@t s vhich record the fre.w number, the date, and a val;iety of other data necessary inthe intcrpre$ation of t`ne pictures. . . l3ocv.htion of large agar-Pilled glass trays or of trays~ petri d2shes is no:? accoxpliskd by the :usc of a lribrating nozzle ~rhinh generates a strea;l Of very fine droplets con.Lai.ning about 10-y liters of bacterial cucpcnsion per droplet. Uzkder cozptl';;~r control this dis- penser can deposit the droplets at any desired distance thus "plgntinz" the agar with droplets containin,: bactcrla in regular roi!s and colons whicil grow -into regularly spaced colonies. This development "Increases the capacity of Cyclops and fill do the same for the i'incl Ik...brtaitcr as ml.1 es mal;ingr the fir.ding and scan&n;5 of colonies by computer much more ragid and economical. Vith colonies lmm apart ES planted by this microdispenser, the capacity of the Bxbwxiter rdi.11 be about lo3 colonies per batch. Ii' accurate rates of colony gro;ttth and ~s`ccisz observation of colony morphology arc necessary, the colonies xust be kept 3 to 5 mm apart dcpzndin-:: on the circzmst~~w2s.` Bv electrostatically &arg,i_ng the droplets as they are formed, it is possi.ble'tb deflect them in desired patterns and on this basis :re no51 have in operation, a "swth dripper" which speeds LID . the plantin;: olr the trays by*a factor 02 Y.-or more. The usefillness of this ^ dispenser will be increased enormously in the future Ghcn laser li@k-.&tters~.~ from,the individual for:xing.dro~lets &ves a signal that can be Tused to thro:? .way ekpty droplets end droplets containing more than one bacteria by the electro; stat3.c deflection system. It.should then be possible to dc-posit; one and only one ceIL on every site. Contamination problew arc? also minimized by the use of this cierodirpenser because a;ly colony grouting at an "il3_e~sl" site on the agar is automa-kkally rejected as not part of the deliberatelgr inoculated system. (j) Auto~atk Film Processor. An autowtic f3.131 processor has been installed"' and modii'kd -Lo develop OL r XLm to a gax~~a of one in a very reproducible and cconom-ical'.T?ay ss that o!~ly mir,w corrections lneed'to be ma& to give'reliable optical density measurements 01 images on the filri. (k) A larze caps&y . sir sterilise$ has been built, tested znd is providing sterile air rcquireci for a. ntu::ber of pieces 01 equipment for Cyclops. (i) Washin? glrcs .*' trays is no8 accom@i.shed by the use .of an old comrxrcial; laboratory &d. - sswa rc wshirl- -ti machine renovated and modified for use tlith thr! Qclops project. ,. Privif.e~ed Communication . ._-.___. ._... ,__- _.___.._ _L _.-._ -__- .-._ .I-.II..-m_- . ._.-w v-...- --.. . -. .,`, . . . . . `. . -PDP-G, film sccnncr, the PDP-3 coqzter posi,tions the colonies..of. interest under the Picker llhich picks up the first ItOO colonies -of interest: These can then be replicated on fr;ezh a&% 'of various composition 0; can be streaked put ,for . mutant purification by noving one row. 02, 20 quart 8 roes at a time across the surface of ciTrcsh agar. After rapid heat sterilization,.an~bher 400 colonies can be picked. The n&v Picker and its computer to be in operation in Eccmbcr 1973, (n) .!Pray-Vashin.~ Bchrine. About half the size tray+rsting r;zcbine accepts stacks of 64 trays of the Du.mbr:aiter itse.lf, the from a noveable magazine, strips off the agar 09 one frar:~c at a time, wxhes the frak and returns it to the bot- controls 8nd.programs is expected tom of the stack. Construction of the tray-washing machine Is under way and is.eqected.t&be complz ted duriq January 3.5)'$ except for plumbin,rr and electri- cal interconnections wkth the rdst of the system. (0) Dwbwiter. Thk design of the Dumbwaiter itcel.f is t&El. under wy and the ~construction of the.nachLnc i s expected to be ooxplete by January 1375. Since all of the more soplzisticaQx3 portions of the W&xaiter design have already been tested on the Cyclops system we expect to have the Duzbuaiter system fully operational wcll.before June. 1975 including all or +hc ancillary facilities many of which a.re in constant use already. 3. IMa Analysis. (a) Flying;-spot scanner. The function of.the flying-spot scan`ner is analogous to that of a tel~visicn cr,in`t-ra but is baacd,on the-use of a precS.siDn cathode . xay f;ribe on which a-t&y spot.of light (less than 0.001 in&s in diemeter) can be inktructcd to appear on anypart of the race of t$c cathode ray tube trlith an .sm,rracg of 1 part in 8,OOO:alon~ both ::;and y axes. A hf&&ality fens thrarln an -image of this small spot of light on a i'rsae of 35 mm plus-x film -&kid a phot~~ultipl~e~ behind the-filr.1 measures the ar;ount of 1iGht that passes throwh the film at that particxlar point. Under.computer ctntrol the spot can carry out an orderly raster scan of the L!ag:c rlith a resolution 0-I 8,000 liner; or it can carry out particular geometric strategies to outline the boundaries of a particular object. TIae scanner is under direct control of.thc FDP-6 cO2lpute~ with high spezd interface B constructed in our laboratory. It is able to scan the entire pzcturc wit%\ a resolutio.rl of 3,000 lines and measure the optical -density of thz film vith a precision of, 1 pzrt in 64. The first step in a&$- .zinG the picture is to locate all the qbjacts; determine ?thich of t&m are. round enokh to be considc-red sin@.e colonies; and cor@ute the center and the diameter oZ' each colony for use in calcul&in,rr gro~~th rates of colonies : (distmeter as a function of time of &rol&h). For experiments requiritis recogni- tion o$ colony morphology, the comt$tcr nexi; instructs'the flyinG spot to pass slo~~ly'across the diameter of the colony, m:a!=i!ig an op%ical density ncasurement at eveq step. T"is results in 300 or kO0 optical density measurements being made per diax~eter.. The operation is repeated for four diawters and.averaged to eive a diametral optical density profile for the cola-ny. This profile is . then used to determine the diameter, the highest peak height, and Fourier knalysis of the shape is carried out. with as many as 15 Fourier coefficients being recorded; To include features such as pQmcntation, surface iridescence aed sheen, turbidity due to.small optical inhomogenieties, and other subtle but ;rist&Uy oi>serv,-hle proycrtiee, ric often take as many 3-6 five,differcnt i~laci; r.j:$. yh:i, ;,2 p;;s~~y7:;. -,j~lc al;' each ~Qli! 0:; ~.+-z:.,; L?c:<,P,I; fiyc di~f::l+c!lk cO,lCX xl:tc~!r . 5-1 i: blue -:,;c:: `:-@'~P"'::l"c 2.3 'oft>rl 3.:;i2:,:;qtic, 1:2m:xe i;lxcf lj.@C is - 21 - DGnal4 A. Glascr .._., C' .`_ . .scat.tercd more L-y ecall tti;';sid re&.crns than Ycd li&t evcns aYe avzilrble. Wncc each photb~rcph nox has the image of an optical gray vcdge in it, the ~ckr\,ricr is able to mcttsure the densities of the images of these steps on the 3 5 m fiti and $lalre corrcctiops if neces`sa,ry to i-ts optical density scale and dctexmine . A. GlG.~er, "Control of initiation of D%I synthesis in E.' c.oli B/r," Pkoc. I;z%. .&cad. Sci. 62, 255-262 (1870). I C. B. Vard and D. A. Glascr, "Cokh.~i.on betxecn rate of cell Growth and mte of DZA synthzsis in Eschcrichia coli B/r," Proc. Bat. Acad. Sci. E, lO@.-I.064 (1971)* .*. . : D.'A.,.Glascr and C. B. Vard, "Coxputer identification of bacteria by colony norpholo&; Frontiers of Pattern Reoo,Tnition, Acad. Press, 1~. Y.. (1972). 3. Couch, J. B.x.-?: , D. A. GlasW, 5. of bacterial strains by analysis of International ConLfress of Genetics, J. Raymond,'J,- Couch,' D.-A. C&s&,' Raymond, .and T, Vehr, "Autonated recognition .colony morphology", Promedilqs of the 13-;;h Berkeley, Calffornia, Aqust 1973. (Abstract) and'C. T . bkhr , "Autom';ic &.edtion~of '. condjtionally dcfactri.T~c nutar;ts of nicroor~anisns," Proceedings of the 12th I~-Lermtl;om.l Con~xsa of Cemtics, Bcrkclcy, CalFfornia, A~qust+ 3;973. (Abstract). C. T: Vehr, L. !?aSliell and D. A, Gkser, "Isolation and characterization of cold- sensitive D??A mutants of ?Ischerici& coli KX?", Proceedings of the 13th Lntcrna- tional Congress of Genetics, Beri;cLey, C&ifornia, August 1373. (Abstract) ( In addition, biological r;ork supported by GM 12524 (now GH 13439) that has been repOrted in the same period, and T&II., rzotivatc som of the first applications,of the automtion systein are: . . . . 1. P. Scotti, "!lJx behavior of tmperature-,, =wsitive T$ a?%. polymeiase mutants in ., temperature shift experixxznts", virology 4j, $6 (1970)` c. 2` 3* . 4, 5. 1-l. Rane, "Sam effects of nalidixic acid on conjugation in Escherichia coli Kl2", J. Bact. z, 45-56 (1971). - . L 'C. `B. Vard and D. A. GIaser, "fnhibition'of initiation of DRA synthcsis'by 10~1' concentra%ions of penicillin", _f R: 14. Burger, "Kinetics of kbelirq of fast-renaturing D3A in Bacillun subtilis", 3. &@l. :Biol. 2, 1~3-31 (1971). t ~,r?p+;fyf.:- T,, -' *i " - 2.3 - Dmald .A f. Gb s.q r-I_ Privileged Cozimuuication . I -.._-. me . . _. --.._ -*.. . .- . ..-. . ..--.- .-. . . .._ -..----^*. . .- _I.* +.--.----- -6. R. I& BurCer'and D. A. Glascr,. "EEed2 of nalidixic. acid on DRA replic,ation by tolucne-treated Escherhchia .culi", ,. Foe. Rat. Acad;.Sc+ 2, 1955 (1973). . . . .7: D. L. Parker and D. A. Glaser, '%hmrmso;ial sites of DU&r,lcmbrane attachment in Escherich2.a coli", submitted to J. Mol. Biol. September 1973. . . 8, D. L. Parker and D. A. Glaser, "Rffect of groxth conditions in IN'A-membrane attachment in Escherichia coli," in preparation. . ., .' . . 9. A. H; Dougan and D. A..Glaser, "Rates of chain elongation'df ribosomal'.'RN~ molecules in Escherichia coli", submitted to J. &!ol..Biol. 1973, . 10. L. Was?rclf. and D. A. Gla&?r, "The iiolation and partial characterization-tif mutants of 3:. &li T?ith cold-sensitive smkhesis cxz INA", in preparation. I A. 4e. Staffing W. K&th Hadlcy 1 Assistant Professor of Clinical Pathology and*Laboratory Kedicine, UC`I&dical Center. Calvin Vard Postdoctoral F~llotl 1967-69 Assistant .Research Biologist 3.9~9-71 -Beverly Wolf Assistant Research Biologist . + Ronald Dakcr John Bcrcovitz James Bcrk Fraser BonncU John Couch Ted Fujita Robert Henry Lcif Hansen Lzrry Johnson Alex Para Associate Development-EnGrg. Assistant Development Eqrg. Associate-'Devclopmsnt &Erg. Principal Prozrammar . Rescarch,Associate ' Assistant Development kgrg. Senior Deveiopment RnSrC. Principal Development Energ. Associate- DcVClOpIIIeiTt lBe;rg. Asoistant Development Enf~rg. 1970.71 1965-72 &4-*resent Z/73-present 3/73-pzsent 1965+2esent 1971-present 1969.present X964-present 1965-present 2/73-present g/72-present Privllcged Comm3nication B) Specific Aims; C) Methods - 24 - Glaser, Donald A. :- 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 cdnditions. 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 +s designed to carry out on a.large scale, 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 conv&sations with a number.of scientists and involve a 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 proposed 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. !I-) Isolate, map, agd characterize temnerature sensitive mutants of E, coli unable to synthesize DHA at 2CoC or at 41%. Donald A. Glaser, Professor of Physics and volecular Biology, Untversity of California, Berkeley. Method: Automated replica plating and incubation at the permissive and restric-s t-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 co,mpnter 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 episomal completientation 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 DrJA synthesis. (Dr. William Wickncr in Professor Arthur Kornberg's laboratory, Biochemistry Department, Stanford University, is studying one of our cold-sensitive mutants that may repre- scnt;a new DNA gene). An understanding of this most complex and central process j.n \~,?c",~rj,~. j.s b;u!:J t:, I?? t:':j?!~ri:';:~'~ f:1>: tQ,&+j++r,.: 132 j& 3"'" 1 et? r L.~.--4\>ocz prvx?r;sc.::: in' cc!.lr; of higilcr orC;:~nisms, rinclz&irig prol~.feri! Z17:: anTmaIL cells. AltcrnatTvcly antibiotics that function :-'ji pc:r,i,urbing DZA synthesis m&y be understood or - 25 - G'L&ser, DonaZd A. Privileged Comr,un~cation rationally sotIght if vulnerable features of DNA s$Ith%zsis in pathogens is understood. ?-I Measure anomalous DHA synthesis events for temperature-sensitive mutants, for UY 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 DDA 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. Deleti.ons can be scored as non-reverting auxotrophs, and duplications can be -scored by assiiys for certain enzymes. In particular colonies are able to grow on lactobionate as sole carbon source only if there is a duplicq &tion in the lactose operon. Chlorate resistance is being used as a selective condition for deletion of chlorate genes whenever a nearby site for soze other function is also affected. These and other assays will be used to study the roles of various DXA synthesis-related genes known to affect UV sensitivity, recomb2nation, 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 DIU, duplicating genes required to produce large amounts of product, providing surplus duplicate genes for future mutational expertients, and enlarging the chroponome to provide scope for greater comp2exity. The probability of these changes must be affected by the structure of EI., itature -- Clark, D. J., Cold Spr. Herb. Iiev Biology 24.2, 130 (1373). Symp. Helmstetter, C Biol., 237 823 (1366). Walker, J. R., OL, 95, 1627 (1968). Clark, D. J., 95, 123 (1968). Hirota, Ye A-t Jacob, Fe, Ryder, A.,'Buttin, 35, 175 ?????? o G., and Rakai, T., J. Mol. Bioi. ??? ? o Picrucci, O., and Helmstetter, C. E., Fed. proc., 28> 1755 (1969). . Previc, E., and Richardson, S., J. Bacm797, 416 (1969). Pritchard, R= H., Barth, P. T., and Collins, J., Symp. Sot. Cen. EPiicrobiol., 19,. 263 (1969) - Zusman, D. R., Incxuye, M., and Pardee, A. B., J. Mol. Biol., 69, 119 (1972); Jones, N. C., and Donachie, W. D., Dir., D. E., Nature Nev Bxym, 100 (1973). and Helmstettcr, C. E., J. Bacterial., 115, 786 (1373). Marunouchi, T., and IIesser, W., J:1401. Biol., 78, 211 (1973). Hirota, Y., Ricard, Ii., and Shapiro, 13. In L. A. Maneon (ed.), Biomembrancs, Vol. 2, Plenum Publishing Co., New York, pp. 13-31 (1971). Ricard, M., and Birota, Y., J. Bacterial., 116, 314 (1373). Taylor, A. L., and Trotter, C. D., Bacteriol..Rstr., 36, 504 (1972). LOT,, K. B., Bacterial. Rev., 36, ;8'~1~ Hong, J., Smith, G., and Ames, B. N., Proc. Nat. Eiserling, F. A., Acad. Sci. u. s. 68, 2258 (1971). and Dickson, R. C., Ann. Rev. Biochem., 1~1, 467 (1972). Significance: Detailed understanding`of the relationship between DXA synthesis and cell division in E. coli may give important insights into the same relation- ship for proliferatin S animal cells, which ge.nera1l.y do-not synthesize DNA except in preparation for cell division. .' -_ 5) Studies in biochemical evolution in E. coli and B. subtilis. Joshua LederberG, Professor of Genetics and Biology and C`naiman of the Genetics Dapartment, School of Medicine, Stanford University, Stanford, California. We wish to observe alterations in polypeptidc products resulting from mutations in synthetic genes (generally synthetic ho:l:opola?er sequences) which have been inserted into the genomes of E. coli and B. subtilis bacteria. Imrnunochemical methods Ml1 be used for detecting these alterations by examinin;: 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. 6) Genetic organization of the E. coli chromosome: position of the translocated lactose operon. mutation rate versus map Gordon Edlin, Associate Professor of Genetics, University of California, Davis, California. . The purpose of these experiments is to probe the genetic organization of the E. coli chromosome. Ultimately we would like to understand why genes are locatea particular sites in the chromosome. One approach to this question is to mcaeure frequency of mutations in 3 gene (or genes) l?hri.ch have been trancloccted tc r+ r,Lii)::1; 02 dii"';:?':.:j'> :::i.';;C:; Ifi y::s (~j'.:l:~:;;;'~~.,._, Li r::S2,;21 :;>SkL::: Ii;*, ~,~A><~i~~ c>E- pCil!::tnt:: is tYrOl"i~j.~~ri by- t:?c !.:`c-i,na~ 3P(?r!)::. ;; c-2; zc n."' ';'l;i") irlr Q:py:[c4; T;l?iCll a';'" genetically k&on,? except that the Lactose genes have'been translocated to a .- I . _. Privileged Communication - 29 - Donald A. Glaser _._ number of different sites in the chromosome. These strains will be mutagenized with a variety o f mutagens (nitrosoguanidine, ethylmethane suuonate, 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 pathway, ribosomal-protein, etc. Genetic techniques for constructing ths appropriate bacterial strains already exist. "Enclosed 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 I \ . . . ' . . : We will mutagcnize 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 RX13 lactose agar. Lacf 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 probaS1.y need a dry run. Once that is determined we can run then as fast as time allows. I presume we will work with Phil-on this. Let me knew hot7,and when you want to proceed.N . ._ _.. . ._ 7) Recombination deficient mutants of E. coli. Alvin J. Clark, Professor of Molecular Biology and Bacteriolo,gy and Immunology, University of California, Berkeley. Method: "Our work in large measure stems from the discosrcry of recombination deficient mutants of various recombination prcficient strains of E. coli. In doing the necessary mutant hunts the present bottle-neck is the picking of colonies of survivors ol" mutagenic treatment and patching them in geometric array. I am very interested in testin g the dripper you have invented as a means of depositing cells in ~eor.letricarray prior to testing their clones for reconbina- tlon ~~cf~.c:izx~:. ' Y I-c f. r: -,-2:-., ~-"~."jl>~~<~ f!?!" I-. -- -"-(y--! .Lb,y!';c .-,q L ..' -. r;yi c:rnr!-L~~.n.~r-tc~ '..:A? h--r- ..- ..I _ I-c.\ I. bbcn sj:l;-Lin,< 211 t:Cc:l;;_12 0.: t;:e yk?`rjOr i;,-;rth at 42 (pelmeasc denatured) and no Gro::th at 37 (pcrmease functional) in the presence of the metal. Having Obtained such mutants I rjould test them for susceptibility to metal mutagenesis at both temperatures. I realize my proposal is perfectly straightfonxxd. If I carry it out.with the tiJnc and resources at my disposal, it is at least a year's uorl;. With the "dumbr~~aiter" I think I could haTie the first mutants in several weeks." - _. I J Privileged Communi'cfition .- -... e-b. ..-. ._ - .- -- :--_-.-----.--.--I-- -_-- --- - .-..--e-*.--m-.--.. -----e---w.. Microbiolo~, School of Medicine, Stanfbrd University, St&ford, California. - . . -. i ~. a 1 I I `i i i . : t I i i' I I I 1. i 6) ! ! ! 'For experiments bn the mapping of genes affecting the virulence of &lm&ella. - species, it is eqeciient 7;o obt-, &in Genetically marked sublines in particular strains. For example, in lines of S. typhinuriu,, 7 which differ-from the available genetically marked stocks of strain LTZ by their high virulence for the mouse, on intraperitoncal` itioculation.. It has been. the experience of several.wo&ers that::auxotrophic :nutants obtained bN mutagen treatment of virulent strains of Salmonella coi:li-nonly nave un::anied additional mtitations causing reduced virulen'ie, by unho~:n nechanisms. Therefore, in theory, the best methjd of procedure would be to introduce chosen negative alleles, dctermininz nutritional' requirements or.inability io ferment pnrtiwlar substrates, by transduction. To do this by ordinarily av?iJqble methods, even :jith the aid of penicillin enrichment, is hardly pl-acticable, becau::z of the amount of labor required to de'iect the rar'e transductants, Tihich cannot be selected for. Dr. Glaser's a.pparatus should make possible the detection and isolation of the desired transductants by an automated procedure. A second problen, in th: StXle general field, ic the isolation of par- 'titular classes of cluxotropiric, etc. Cutant in mouse-virulent strains, for possible use as live vaccines, stably con-virulent because of, for instance, gros?th factor ' requirement, but otherwise unaltered. Nutants blocked in the synthesis of the [ dizminopimelic acid component 02 t'ne ha eterial cell s:all should be unable to mul- ' tiply in the tis:? Les of a mm..?.?alian host because of absence of this substance, a component of bacterial but ; not of eukzryotic orzanisns. Heavy mutagen treatment of the bacterial strain to be 'used would be likely to cause additional, wwsnted ' mutations: furthermore, it is unlikely that.such mutants can be selected for by the penicillin enrichment technique. Probably the only way to isolite such ' mutants is by direct exarsination of a bacterial population for cells able to produce small colonies on defined medium supplemented ?qith a small amount of dixJino+elic acid and able co resuiLe gror.$h on prolpi-5 Z-o~ of additional.diamino- pir;elic acid. Dr. Glaser's cethods and apparatus should make this feasible, where-.' as it is.hardly so by other methods. \ Proline non-utilizing mutants of S&xonella typhiauriun. I --- John R. Roth, Associate Professor of Kolecuiar Biology, University of California, ! Berkeley ! Ide've been ana1ysi.q.the proline degradative pathway. It involves an operon containing three genes, Permeasd mutants two genes for degradative enqlfles and one pesmease. can be obtained by'positivc solution. The other two classes we more difficult to obtain. on proline, Because even wild type cells grow rather slowl) the standard peilicillin enrich!:lent works very poorly. Screening of mutagenizcd cells with your apparatus should perlnit mutant isolation. ThCSe will bo strains which fail to grow on proline as sole N. source but can use - L either VII, or glutamate as a nitrogen source. II 431 j . I * 14) I Isolate mutants temperatures. Isolate mutants Of of Bacillus subtilis deficient in DI?A synthesis Bacillus s:btilis resistant to certain phage like p. hydroxyphenylazour~cil for studies on DXA syn+hesis. and to drugs A. Ti Gznenan, Professor of Genetics, Stinford University, School of I&dicine, ! p:... ) -. _ . . .._I , . " . ~)uit,`li'Ji it, (.!9.&.1:~ 0:`;:i.X. \ at high or at low : -._-- Our research project involves the study of th2 mechanism of lNA replication and its genetic dontrol in EnciXLus subtilist a trans?omablc bacteria. lie have isolated several temper?kure scnsitivc mutants t'nat are defective in DXA synthesis The themoscnsitivc pkotcin ban pecn studied in a feM cases. There are abbut 9 groups of genes that control DXA synthesis. 'Fncke may be even.oore. We isolate these mutants routinely by conventional, slog and laborious procedures. The automated petri dish machine would be idcal for the above project. 17e are speci- fically interested in both lov and high teqyeraturc sensitive mutants, and mutants th+ rarc resistant to drups like p. hydro~~~henylazo~acil. This drug specifi- cally inhibits ?XA polymerasc III in Bacillus subtilis. Polyxerase III is dQ?ectl involved in DXA synthesis. Resistant mutants ~o:zld help to locate the position of the gene for the enzlxe. The system is also adaptable to test phage mutants which are cwrently studied. The instruxnt is a very valuable and unique tool for our projects. ble Tcould very much like to collaborate with Dr. Glaser in obtaining several importent mutant s projcc%s in cell bioloa. and adapting the machine for other related . Screening for possible mutagens camong environmental chemicals by mutations affecting sporulation in Bacillus subtilir. , Lwrcnce E. Sacks; Research E.crobiol~ and / i Jees T. MacGregor, Research PlxtrmEcologist United States Ikpartnent of Agriculture, Agricultural Research Service?, ~ i Western pegional Laboratory, Berkeley, California. . 1 Thousands of chemicals, whose biological effects are little understood, have been disseminated into our environment and the food we eat by modern tec'hnological society. Host frightening of these chemicals are the mutagens, tlith their poten- tial for teratoscnic effects, cmcer, and unkno5zn'l&g-ter.3 effects of alteraticn 1 of germ-cells. . In screening for possible nuiagenic chel:lic&, microorganism systems offer the : advantages of speed, sir;lplicity, and economy over anix systems. A disadvantage of microbial systems noI? in use is that they test only for mutations occurring in one or a fell genes. A bacterial system sensitive to inutations on many genes, scattered throlshout the chromosome, rlould seem to offer imporiznt advantages over currently used systems (1). \?e believe such a system is that governing sporu- : lation in the genus Bscillus. Sporulation is a very complex process requiring the participation of a minkxum of 28 ~?crons 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 (3). : . . c Selection of azporogcnic nutarks is simplified dy thefr chakctcristic. white color easily distinguished rro,? the Tlild-typz brown colonies, colored by formation of a piment late in the gporulation of B. subt?.lis,Ihrburg 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 unrzvel ,the genetic control of s$orLLation. I Ve propose only to invert this procedure, and to use sporulation mutants to identify ner: r,;uta~enic agents. , Dr. Glascr's instrwent capable -of identifying --._- 9 sin&e mutants in huge populations, will be of great value in idcntifyin 3 mutagenic activity at venr 10~ concentra- tion levels. This coxbincd use of a bacterial s 7 :,stcn involving over a hundred , genes with scanning by an instrlwcnt 1.3 -7 cspnbl c of ;-den-l;ifyinT mutation rates below ' .x ::?:s::2.. .:. r-!::i.;.?.-'; \ i;:l 3 -,: -: '?'? .r . . ,, * ,- ", -. '..-A; ,.-. _. ' -., ..-.. . . 3 '(' . . . . . . ..-... ,_ _ . . c 2 .d- .: - r c; ." .i. I ;__, ,i.. ._ 1... .~,.';~,:~,~~ J-1?;? * < (?,p;l;~.i';'~~?lf- .-I xc'iv;~j:-'ntc: c~IK~;,.-~.c~.~!s. ,. ; . . .._. _ .- ._ _ - ._. .- 4 .- ,. .`r:!;, ;`:.!:y -;`y. - 33 - _ LAmLJf .A., crlaser ., ___ ---r ._- __.. . . : ' . . --. i t : t -. *. . _- l?ri;~fl~~ed. Co9xmicatior! ' -- - _.._.., v.. .__ ______ ._-._. .-- .-^-__. . _._ . . ..-.- __. ___-_ -..-'_.._____._ _ L_.--._-C-----.- -. - We smmrize below som adva.ntaSes of'thc proposed system: - . . -1. It is based on forr:z.rd nutction, the, nest general type uf detection systcn. Any type of mutation which ixxtiviat3s or substc~tially alters a gene essen- tial for sporulation will be detected. 2. A large nuxber of genes are involved in sporulation -(2, 3). Sane mutagens : are specific for partic* regioas of the EIA.. Tie more genes surveyed, the . . less chance of excluding cutagenic "hot-spots". I 3. d Sporulation mutmts are often < : ch eracterized by a block at a particular staSe in their morpholo&cal developmnt. The frequency of occurrence .of particular stages of arrest TliU.pemit an .gssessment of the randomness (or specificity) : of each mtagen. . .-' I 4. The B. subtil' LC Systen is well-suited to genetic studies. Righly transfoma- blc strains exist asd nany genes have akeedy been napped (:I). scarming -cystem, `however, is not linitnd to the Di~~,e:lted B. IFS. Glascr's : subtilis colo- . riles.. Other r$ell-studied species (e.g. B. neza.t&ium, B. coreus) nay also be i employed. _ I References . 1. Hollaender, A. (Ed.) (1971). Cheraiccl Nuta~ens. Principles and Methods for I Their lktection. Plenum Press, I$. Y. Vol. I, II, III (1973). . 2. Pig@ot, I?. J. (1973). "bkppi~g of asporo,~onous nut&ions of Bacillus i suhtili z : sc",erFol . A nlr,ir~~m estimte of the nmber 02, sporulation oper3:iSF'. 1114:12&i-53. I ` . 3. Balassa, G. (1971). "The Genetic control of sp& for,?atioc in Bas will be-obtained lrhich are rxqosonic. for a large but ' 'poorly marked .chromo~ox. yncse strains are diploid for all Gut bne ch'r'omoeokc. A hunt .for temperature-sensi,tLvc mutants should yield nutants carrying lesions in ! the chromosome for vhich these strains are hagloid. Determining the nuiiber of genes involves is fairly !!&cse can then be analyscd. i easy because of the sirxplicity ; of yeast coi:plc1:~entation tests. I'd like to try this in & year or so after I've ! gotten back from satkatical leave. time.) (I'll be doing yeast genetics during that,,. j 1 Genetic mapsing iii EkCChnrOEiycCs cerevisiae Robert K. Nortixr, Pr~Pcssor and Chairman of ?&dical physics &patnent, Univer- ! sity or CalFfoYnia, Berkeley. i I The availability of detailed genetic maps is an important component in detenzining the suitability of an organism for genetic and molecular studies. For a number of years, T)Z have carried on a progrsJn of genetic mzF?ing in the yeast Saccharo- myccs ccxvisiae as an adjunct to ow other studies. These mapping studies r,evc resulted in a genetic na? which c--"-q Gb,,blishcs the location o-i' more than 150 genes or.' 17 chro:noeor.les. Ho~!cvcr, bccausc of the largz nurr,ber of chronoso~~es and the hi,+ ! _ frequency o-" I zcnetic rccor.:bination in yeast, very few heavily mapped regions arc availaLle. Such regions for studies r,e ;!ish to carry out on E;;enz ; ccc import3nt con-version and its relaticn to rcchanisms of i genetic reco&Iination. We believe ; the instrument dctrelopzd by F'rof?ssor C-laser could help to speed up the further 1 dcvclopxnt of gwtic ~11;~ in t?lis drganL&. The'a~pro2ch :.;c prop332 to use is based on the randon cpcrc tcchniqx i describF4 in our-recent r.?ap_ning paper (1?3,--t4- ' mer and Hwthornc, Gznctizs 71:: jj-$. (1973). A Eerier of strains that each carry, one of a set oi' cchre eu~~2xSox in cor.;bic!kion with 1. svpnressible canavanine _. resistance gene and an ad.ciitional selection of nutritional genes bill BE crossed ' to a large series of txnperaturc sensitive lcthalc. !i%e resultant crosses r~:ill be' spor6lated, and the asci iJill then be treated l.!ith @usu?~~c follo~lcd by sonic=- Mon. The sonicrtcd suspcneion ~Sll be inoculated onto complete medPAq containing caixvaninc. Only s'ores LxkinS the supprccsor and carrykg the resistance gene will pov . "drop:out" These cm then be transferred by replica Elating to a series of j plates 'Lo score the nutritional gents and io a "215~" environxnt to ! score the conditional Genes. The patterns of growth: nongror;th on these various media can then be recorded autocatically by the scanner and the resultant data -' j : analyzed for linkage by a suitable corqxzter grogram. In .this procedure it ~i.11 be necessary t0 inoculate at a concentration that reduces ko a lox level the _uroba- bility of clones developing from more than a single sport. The instrwxnt should e;reatl:- facilitate rar?dorn cI)ore analysis both by permitting larger saE:ples to be analyzed and by aEt%xtically recording and acalyzing the results. - -. . .I_ a (..y;!;.>;.,,yii \ , . . .,.' - 35 - . - .- -.- ---- - ,__- F,riv!,lc-cd Ct?w.xc.icnSi@n ..- .._.... 4--.. - .- ,-. w; .- .-.---__ ..-- ---. -. .-... -.-. -.----.- -..----.-. .____ ;___,____,_.. s+v!!c@es and O~~CTiLI rationale may be carried over to address such seemin& { unrclatc(l Thor.@ ccn"Y~a.1 problexa a..s chrcmosome noirdisjunction,bor screcninC, : rxtaCcns, carc.inoC?ns, funSicidr;s and antibFoCics.fpr their genetic effectst - I. Gene conversion end recombinationin unselected nitotic yeast cells ' ,Our current understandin?g of intreCenic reco,mbination in cells committed. to a j ' mitotic cycle emerges from data Generated by selective methods. In effect, these: depend on appropriate sigxzl devices that lead to the detection and recovery:of only wild type or pro-totrophic recombinants. Ho:~ever, we have recently'demon- stratcd the occurrcncc .of nitotic .co-cwxercion in hybrids marked by three of : ; four heterozyCous sites in a single%.structursl gene? and it must be emphasized that multisite conversions do not -&ically C-;cneratc wild type recombinants. r i Thus, thou:yh do-co:nxraions sight represent the most frequent event class, they : remain undetected and unscore,d in conventional selective procedures. By analogy to our studies on unselected co:.$lete mcfotic tetrads, ;je propose to analyze (in , the same hybrid.?) an ~wselectcd rJogulation of citotic cells for all conversional events f,-,lliriS T.:i$L ?in a defined genetic region. ; I Nitgtic gent_ conversion in ye,, 10' to 10-2. "ci; occurs with an everaFe frequency of the order Accordingly, collcctinS a sample of 19- or lo3 u celected convcr- i sional events in-iolvcs screenin:: a total g+clation of 10' &- j - 10 cells, or a i sample beyor,d 'iilc capaJLili+ "y of routine microbioloSice1 methods. Automated I microbiolo,-r equipment, howver, this and rimilar stiUdi0s. * augurs well for the successful completion of i . i . : I. Our anal+ical stratcrJ require:: a) autoxatcd single cell inno&la; b) replica- ! ,b plating tlx derivative clones; d) detecting, locating, c) ir-9 -,diatin,z the rc?lica prints (W or X-ray); i and ,-rctrievi.nS sectored clone-e; e) finally, complete genetic diagnosis of each sectored clone b? random spore or tetrad analysis of i each segxnt. -1 i II. Post-xiotic sc(yre,Ta.tion and- hctcrodnplcx DTT-4. ! 8 .i Common to all molecular models seekinS to account for genetic recombination are ' enzymatically mediated steps that eventuate in heteroduplex or hybrid DiiTA produc- tion. At the in viva Scnctic level, the p--p= ,.c,;cncc of unresolved heteroduplex Di?A 1 is detected by post-rxiotic scCreCa.tion (FYS). F1.3 is comparatively frequent ." ; a.monC thz total aberrant octads 02 Ascobolus or Sordaria. Hol,ever: technical difficulties Hith these forms,.includin~ a paucity of Ccnetic markers, p reclude total and critical analysis. Vith autozatcd InicrobioloSical procedures adapt4 ; to randoll spore or tetrad analysis bnsed on diploid yeasts suitabbd marked T?i';ih I 'i-10 hetcrozygous eitcn (i.e., loci and alleles of I-now? neiotic conversion frequencies), ue could readily assess the frequency, extent and distribution of : heterodulGx DEA in the yeast Ccnome on a statistically reliable base. Sectored : ascospor~z. clones, o-i;lzx5cc concordant for all segr zcating mar-lrcrs -r&U. be i considercc? as I%5 cvmts . i i -z- - Also, from the dis'xibution of KS events among spores produced by heteroallelic i diploid:: of the type -H/L? or 1+/i-2 (notation as before) where the mutant allele : pairs may be chosen from extensive fine structure naps to represent a range of i genetic f.l:i st:1nccs, tlln rc:;ul.arltj.cs r: y:$ b::cic aktrlbut~c of hetcroduplex DM - I j yz 1;;'; :; '..i -!:, : ) ,;.?i...: CL:.:: .-.-_.lO.? ;:r. : L':,C:;,,.I. `*`.::.E1 : l:[;:.1. 1 ::<- :~r!~?q~rp<. `!- . .a- . _. ___. .._._ . . . ,, . . . ..-...- . . . .._.. --., _- ' > .. c , f3;*iie !:I.* :i ?,* 2 ,.I.. . . Donald A.. GJ-wr. __-._ __. - - 35a - - ___-__ Privileged Commun@ation . .- ._C .-._. _hd -..._-__ --_-_-.- ..--_ ; 2&j'- Mammalian somatic cell genetics. se- I... . . . . --.: _-.___ __ _ _____ Frank Ruddle, Professor of Biology and Human Genetics, Department of 2iology, Yale,Un$versity, New Haven, Connecticut. : . , . . . . . . . ._ . I believe that your machine has particular possibilities with regard to the recovery of conditional temperature sensitive mutants in tissue culture : populations. As we ha+e previously discussed, it would seem possible to esta- blish colonies 'in the machine and then to shift to higher temperature and I examine the colonies for retardation in the rate of increase of colony size. It would be possible to maintain the cultures at 34'C as a permissive condit : tion and then to increase the temperature to 38.5' for 3 hr. periods out of t a total period of 24 hrs. and carry this regimen forward for a period of one I or two weeks. It would seem-to me$hat 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- ' l&ion and characterization of temperature sensitive mutants uill, 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 i cells to progress orderly through the cell cycle. All of these mutants can be analyzed by genetic complementation tests involving cell hybridization and j I . 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 j recovered. t . I It seemed to me that your machine could be adapted also for recovery of ! . mutants indiffere,ntiated cells. _ Quite a number of tissue culture cell lines j which express specific differentiated traits are now available. For example, .i we are growing hepatoma cell lines which produce albumin. The albumin is secreted into the medium at high levels. ;jct would seem to be possible-to j 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 i ,. excellent method for picking up non-producers. These cannot at the present t time be enriched by selection techniques. One could also test for reversion j t0 capacity to produce the differentiated product using the non-producing f mutant as the base population. This kind of procedure could be adapted to cell lines which produce hemoglobin, myocin, nerve specific protein, etc. -5 * i !- . . When your machine'is sufficiently developed to make use of mammalian I cell populations ,. I would very much li;ke 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 : j connedtion. - . j . . 1 : . L . . '. 8 . 1. i . : ) ! L *d . . . i . 1 1. i ._ .:. . . i . I . . i -. . D0W.g. A* Glaser., ___. . .---__ --:..-`-._..__I: ____.___ --._ -._- - p, Isolat, 0 and characterize a larCe number of'steroid- and cyclic &@-resistant . : 2. .clones of mouse~~mph3ma cells: . . . i _..~ ~I. Cordon 14. Tomkind, Professor of Biochemistry, University of California, San I - -- -_ Francisco. _ . I.., :: t,. i !' . . k.' . .' -:.. ; *. . M.' . . . . . i .- . ..! I .C. .- c 1' ;-? . .L : : i-3 . , 5 `.. t 1 . .i. . . : : -`, v..... -1' . `.. f. -2 ..:.. : ., \ I.,. "i ., . I __, :- .w.. General &jectives: For some years our laboratory has been studying bioloSica1 i -= regulatory mechanisms in culhurcd mammalian cells. 1.7~ have concentrated primarily on the action of the steroid hormoiles but more recently have become interested in , the: cyclic nuclcotidcs as well. The bul!c of OUT work heretofor has been a bio- chemical andlysis of the molecular mechanisms of cell-hormone interaction. ( Quite recently, howver, :?e have begun to explore Ccnetic techniques to lxx.sue our ob- ' jectives. For this purpose ve havc.been using cultured mouse iynpl:a.~a cells xhichi are killed on prolonged.exposure to either the adrenal Clucocortic~iGs or to cyclic Am?. Thic rea$kc occurs at _Dhysiological lel:cls of the cffcctor~~~olecules and ' presumably reflects the ~11 knotfn irxnunos*upre::si-fe action of the &lucocorticoids ' and of agents which elicit cyclic nucleotide synthesis. In any event; :le have beer. able to select variant 1 ;I;?.phoma cells resistant to the killing actions of the steroids, cyclic I\rZF or both aecnts. Our results to date indicate that the -tre.nsi~ tions from effector-sensitive to effect r-resistant occur at randorr. at a rate, in.the case of the steroids, of 3 x lo- E per ceil pzr generation and for cyclic i AMP, of approx~kiately 1 ::* 10-T per cell p3r generation. Various'nutagens increase the frequency of steroid rcsist~nt cells. Biochcxical anal;rris of ths phenotypes ' Of steroid- and cyclic N-P-resistance had indicated that in the former case, tkiree types of' variants can be isolated: those lackinS the normal cytoplasmi:: steroid binding activity; those where binding tckcs place, but in l.:hich the receptor- . steroid coqlex is not translocatcd to the nucleus; and finally those in which binding and translocation occur but cell death does not result. PrelLk.nary investigations . sqgest that various piienotyp2s also give rise to cyclic AIP rcsistancc. To date ::e have studied or&J cells in which the%yclic nucleotidc binding protein and its associated kinase arc deficient. Specific Aims: -- 1. To isolate a large number of steroid- and cyclic &@-resistant clones of 2'. lymphoxa cells. To determine the frequency of their occurrence and the effects of a variety of natural and artificial mutagens on the transition fron sensitivity to resisknce. '? 3. To determine the biochemical bases of cell killing. 4. To characterize the.phenotypes in terms of krious lcnorgn steps in hormone action. . 6. . . 7- 8. To carry out complcmentation analyses using cell hybridization techniques to determine the number of niochemical steps involved in cell-hormone interaction. . To determine whether the transitions result from ,-enetic or other types of stochastic, heritable variations such, for example, as night occur during ihe differentiation process. TO investi@x possible relationships bctT,een resistance to the steroids and to tk cyclic nucleotidcs. TO apply similar methods to circul&inS malignant cells in pxtisnts with lymphoma or leukemia in an attempt to d,esiCn more. rational therapies for these diseases. -.Donalcl A. Gl.ascr...--....Ll- ._ _ Pr~Ail-~G$ Communication e - .-.- . a__ .____.____ _-_ _._ -..I--- .__- ._ ._..a..C_..---- ____^ .-._ _-..__I.-_.-C..d. _- . .._. I. .' clihical metlicin'z. The &cocorticoids are major'therapeutic agents in'leukcmia. .a.nd in other sali~nsncies. Their eYfectivener,s is 1imited.onl.y by the emergence Of li3?1";.l~Ie Bien . -csistant cell populations. Our observations ?iith cultured cells can' -therefore :~n-e',.~ 9" a useful :~o:lcl for studying hoT? it might be' averted. !lhi findin,: that certain mutagens, in particular alkylating agents, ehnce the con- version from steroid-sensitivity to steroid-resistance already indicates that I 'therapeutic regimes ::hich employ allzylatin S aGents.together with steroids might be: redesiGned to avoid the-possibility that steroid-resistant cells are produced in the'course of therapy. < j t !Lllese studies also suggest that new classes 02 a.gents, ~ucli as the cyclic .nuclco-j tides or c~~mpounds .T: hich elicit their production, might be used in tumor chemo- theracy. The np~>arcntly loxler'frequency of resistance to cyclic nucleotides holds: out thz liopc't!;,at these agents could be more effective therapeutically than the i steroids. . . . , - 1 From a theoretical point of vie?,?, these e-qcrixents could provide novel approaches; to investigations .ool drug and hormone action by combinirq genetics, yjith cell biology and biochemistry. It should, for c;;amplc, be possible to isolate cyclic ' AHP-resistant-variants in ??hich adrnyl cyclase or various specific membrane reccp-' tars are deleted rxking it possible to study.the in terrektion between the e1encnt.k in this important reGulatory.circuit. 9%~ same considerations hold true for the i steroid hormones and studies on their mechanism of action. i . i Steroid and cyclic !MP-resistance! are the result of changes in structure of the ' receptors. .Since these noleculr3s have been identified , md to some extent; puri- 1 fied, the Seneration of resistant mutants can be correlated with altered molecules: Therefore a more complete genetic analysis can be carried out than if the selec- tive i c!:er (e.g. drug resistcncc) were not;,correlated `with a known protein. -5. - -.. I LZnkaSe analysis in mammals by somatic cell genetics. f Theodore T. Puck, Director, Institute for Cancer Research; Professor of Biophysics' and Genetics, Eleanor Roosevelt Institute for Cancer Research, University-of Colorado.Nedical Center, Denver, Colorado. Preliminary discuss ion of this project ha s indicated the Great labor of isolating mutants and,establish& linkage. Feasibility studies need to be carried out before real research plans can be made. Because the Genetic exchange system is"' so inconvenient compared l?ith E. coli, the automation may be even more valuable for animal 'cells than for bacteria.. Sensitive detection of mutagen nesis by changes in colony morphology-ex&ension to additional bacterial and eukryotic cells. D. A. Glaser. b&hod: ,. Since colony norpholo-, 3' is a highly polygenic characteristiti, it should be avery sensitive detector of mutagenesis. Extremely uniform reproducible culi;ure conditions are required to Guarantee reproducible colony morp?lolo.D even in the absence of mutations. 1011 "cxj?Osu1~es", For measuring Gross mutagenic effects do:!n to very we plan to explore the limits of' colony reproducibiliQ for a varj.ety 02 organisms. , S:i-pj:. * ":cancc? : -r. b --. *. . - . .LI :;;,.ccc r: t-Y& - L . . . . ---- COlDi*~J! 2ET!~~!Ol.O[;~~ ?h2fi~cS JTYO-`T3.ti~ 3 2i'ki?7.! O?* p.y::-`p.";:`,`; ],.lli:Ys~~~:!~;~< C &J",;(:<:: Or; *y 1:;7<..3 ;-;>?*j.y::y o:T <:i,()r.7Jj]eC ccl!.?, :"re!l i1.T l;e.[,-;le . / `. 1 . . . ..I . . , . . : . L? . . :: `.. -., .; .- L , `I . . ,Y :. *I ;a. _. > ,: .I ,- . r i. `-1 :\ ; . . ,. I ,- - ,- ..4,: .:`,:`-: .:-ii;<: - 38 - ._ Donald A. Glaeer ' . - .- - .- ._-- - .._ ._ _ _ . .- _ -- _ _ Xkivile~;ed Cor,Uunic;ltion . ..- . . .--.._ .-- . ----- .-_- - . - .--_-..- - ..___-...-..._ -...-_-_ i- _______.__ .I-.- ..-.--- - .-.__ ~ ._.__._ or no genetic inforzztion is availchle; Screening of chenicai and physical t mutagens is an Dbvious appli,ca$~on. i . . ._ . f - Transfox?r;ation' zzd mutation of IV&l?aiian Cells in vitro by.10~ do&s or m&g&s : and ionizing radiation. .i D. A. Glaser . "Tr~nsfonilation of Ikznz1ian Cells in vitro by Lo:? DX&S of XLrays", C. Borek -. /' and,E. J. Hall, %turc ;11!-3, 450-&53'- fibryos of golden hamsters r.,ere q 1 ninced and separated intandividual cells grol!ing on agar. I The cells we& irradiated Vit';l 1 to 6% rads of X-rays, incubated, stained, and the colonies i $ormed (probably about 2 cm in size) . e;rzmined for forms made by transformed i cells. . f * .-' .- Table. of U'fect of Transfor~cd Cells I . Dose Glories Examined Cells Transformed .' i 0 36, ooo 0 : 8 1 .: : . \ 10 17,900 10,200 t j j ,25. : 5,500 8 I t \ I / : i Clearly ljrce numbers of clones were examined for the infrequent event. The abilidy ' - to use larger nLuz?Jers of' cells and e::arzine the clones famed from them wuld make the nwbers found more preci se and allo:? better description of the dose response curve at low doses. . Be4kviors.l Mutants of 14otile Orgagiems D. A. Glaser . c . :: -- - In the original pro>oesl for construction of the DV and scanner system, we de-. scribed possible behavioral studies of motile organicms of standard or "instinc- tive" behavior as llell as- adaptive- or "learned" behavior. The' f&lowing+quoted as an example of the tJTe of study ue would like to pursue sometime during the nc;rl; few years. "Chemotasis by the tants and Analysis 821 (1973). Knolln attractant on azar patterns re.sulting Ikmatode Ceenorhabditis ele:;ans: Identificatidn of Attrac- Of the Response `oy Use oi' 14utants", S. Ward, RJAS 70, 817- --, behavior mutants of this nmatode were put onto gradients of an.! plates covered M.31 egarose beads or scphadex beads.' The dif?ered between the l,ild tsz and the mutants. i i Some studies. : T?ere done to understand the chemotaxis. The hunt for more mutants ws.proposed. Clearly, in hunting for mutants, thz more UOIXYS 1 to be examined the better. The I ,.' voms are pipette. small enou<;h to be inoculated in 0.05 ~1 of liauid from an Eppendorf Tile patterns path can be done bjr are formed quickly and photograph-wll. . : Analysis of the j : co~putcr in the same uay BerG follows the three-dimensional ' : path of E. coli. ., ,i : . ;- Further-Automation Instrumentation Dzvelopm&nt. --- . . __ j .i Althpugh the kmbwaiter and 311 of its ancillary equipwnt is expected to be i .! in full opcrztion Ishen this progrm-progect would begin in June 1975, a number ' : . i f ! .- 1. . 1.`. . :- .-:-.,I: "-(,,' .:o., ( .- 0 :: . . :' -39- . . . Dma.ld A.. Ww?... ._ _ ._. _. _. ._____._ Pl'ivilc:~:E3C?._C_ql'!ii'.U'lica~ion i - ..-. -- .-.. _ -.-. . . - -.. --- .-.- -- __.. -- - _.-.--_-_--. :I:-: ._____ __._ ;.. .__._. _ ..i.- -_ ., i I(a) Optical Cell Sorter--At the present-time thz vibrating nozzle inoculator is ! - used for laying down regular rays of drpplets containing bacterial suspension. . In the futFc it will % used to deposit yeast cells and ahimal'.cells cis well. If the concentration of cells is adjusted so that each. droplet contains on the average 1 cell, then l/e of the droplets vill be .empty, l/e of the'droplets wilX contain 1 cell, and the rest of the droplets trill contain more than 1 cell. i For most measurements the only really useful results come from colrjnies descen- 1 ded from a single cell and the empty droplets are of obviously no use for most t measurements. By illuminating the .droplet at the time of its formation la&r : light, it is possible to make dark field measurements of light scattering, : color, and fluorescence, which si[l;nel the presence .of a cell and give some i information about it. Such instr+Tients work well with animal cells, but requir; further dcyelopnent to detect bacteria, which are much smsller. . ..' 1.' .-. W. A. Bonner, H. R. Hulett, R. G. Sweet, and L. A. Herzcnberg; "Fluores- j ! cence activated cell sorting", Rev. Sci. Instruments `r3, 404 (1972). - 2. M. J. Fulcyler, R. B. Glascock, R. D. Hicbert, and N. M. Johnson, "Device which separates minute particles according to electronically sensed volum~",~ Rev. Sci. Instruments 40, 42 (1359). ) , - 1 \ No&',?f the existing systems seems capeble of detecting bacteria and we hope i I to build such a system sensit ive to bacteria as well as to larger cells. : . i Increase Film-Scanning and Computing Speed --Since the DLnbwaiter can easily te!:e one photo grmh per _ second and since the fib-scunnigz time ranges frcn 10 to 20 i seconds per picture depending on the experkxnt, the film-scanninG and computir,:: 1 operations x.411 be rate-limiting steps in the output 02 the entire system. Ue i are, thereTore, veq anxious to cut the ana&yois time !jy installa.tion -02' the PDP-11, PDP-10 scanner coclputer system as &ll as by soze soft:,are imgrovemcnts., Install *levision System --For some future experiments it.1511 undoubtedly be : useful to analyze biological systems iri real time and to int&ene in the expcri- ments without having to wait for the several-hour delay of taking pictures, developing, and znalyzirr~ them. For this purpose YC plnn to inE-tall. e tclevisiori system connected directly to the coxgter which will eliminate photozrzphy. In : addition to allo:Gnz real-ti_re intervention, it Mill be a considerable saving in the cost of photographic materials. On the other hand, the tilevision--system does not have the reliability of experirzents recorded on film, nor do television, cameras have as high resolution & our pr;esent flying-spot scanner. We imagine, therefore, that we :!ill use both systems depend,in g on thc.needs of the expcri;::cn",'. Pradiation E'acility--Vz plan to provide a facility in the DumbT!aiter for irradiating cells i\lith ultraviolet or infrared 1igh-L and also with ionizing radiation on some schedule as required by the expertients. 1 I Semi-micro Photography--For study of very small colonies we 'will need to provi& a semi-micro photosrz?hic system :!hich r:ill photo&aDh a l-cm or even 3-1~0 sc$kre on the agar ineteed of%hc present 10%mm square. Tiere is a trade-off bet-tqezn the time and cost of photograph3 7 and the size of agar area covered. Opt imizinL: the trade-off M.ll. require dif;"erent magnifications for different experiments.` : I:rG c.111. I'?nlli~ll..~..';ic,n D:-.+ :n--Cy: ?)ylyz"v n1l.r !?`-, p;c ';g lx<' c.c! nyi..- -- ALI @CkTZ,, 7-r -';>.;iy.!:,zy . . - -. L. ; 3.:". -;~~."-.-'-:...lj..::5 i]a; ,-l~,:.~+ -.,.::.t;?.:;j.r: .e':,'l* c>].~;.:jez o;y >;. ';r:l.j pr!:; C,.-\.j-- L vi.. cells that have s3xila.r physical properties. We can well imagine thai, 'okher P! I;`:":`,i . . _.. p!'y.%.* .dJ.], .,. :': :;- -' Pr.-, e;: ,*;, ,,l",.?f-:,:..,4- ':..'T.:.A:.dj( ;;T" -y .~:.:?:.:.`,:!,,!I-.',c" ;'" - S;;r'Cr21 C!2!.1, z:r:nQJcl.!l :.:r?:> ,:,;:&&r r.or"4 : : -L 3-l iJ 2 ::2qui.r2~i :?ori Li.:!!:! to 'i-tip: . *. , 1 ~~,,.?:.;i,,J..:i!p? c: "C 40 - Donald A. Glaser . -*, .- ;. ._ .--. .--* .I.. _.__._. _. -----, ::. I ::`. _. `,.`.' . , :..--. ,Exivilcpz.d .Coimxuni.cation.~..,,.. ! --..----..._I t _-- ---_. -.--- _.-___; . . _. i Do Significance . . e-\. -, i . . . . ! t ! Inthe discussions.abovc of the Ix&ti+lar~,biologic~l research projects, ; ficance of each one was pointed out. the signi- In general, these a.oZcations of modern auto- ' ; mation technology coupled with computer-directed pattern recognition and analysis of i data offer a po>?erful new tool for accelerating research in a wide variety of-fields : I of molecular biology and cell biology. ; materials required They reduce enormously the labor, .timc, and .i to isolate rate mut&s critical at a number of stages in research, I as Ijell as to measure iqith high accuracy frequencies of genetic and mutational events : i which must be known for the genetic di ssection of important biological processes.. ' i . 1 I '! I3 addition to the great gain.s esyected in the speed of research in fundamental 1 biolom, the same large -scale autox~tion techniques offer : great promise for a variety! 'i -, of bio-assay applications, including the screening of environmental chemicals for i. i their potential mutational and carcinogenic effects; thctesting of proposed anti- i biotics, antineoplastic agents, and 'cell regulatory substances.' In additional to the j f possibility of large scale testing'of chemical agents, it seems possible to make : ; highly accurate measurements of the effects of ionizing, as Ijell as non-ionizing, ! ; radiation on a variety of clonable cells. The in-forma'iion resulting is imporian'i to i studies in fundamc-ntal biology as T?ell as the difficult problem of setting safe ' 'standards for allowable exposure to' ionizing radiation among the general population ! and among l:orkers in industries involvin g the presence of radioactive substances. ; With these large scale methods, it may be possible to extend the dose-effect relation: ship down to very 10~1 exposures and- so to discover in an over-all sense l:hethcr there 1 is a threshhold or minixum dose belo?: which repair mechanisms prevent any detectable i genetic damage at the single-cell level.: _ . . : I , Finally, the success of these applications of the cutting-edge of modern technology serves as a demonstration which may stimulate siJilar applicaticns in industrial i as l,ell as medical and research sectors. We alr&ady kno:! of scvei+al projetits for strain improvement of antibiotic producing organisms that have been directly.stimu- i lated by'this work. Representatives of a very lar ; ge number of pharmaceuticalmanu- : facturing firms, instrumentation manufacturers, and chemical companies have visited ' our facilities. Suppliers of agar for.medical and research pur_ooses have also visited our facilities and have discussed with us their problc. MS in maintaining uniform repro- ducible quality in t'neir product. Variability is a source of considerable difficulty' in both medical and research applications and we have agreed in a general \?ay.to ; measure batch to batch variations by its effect on colony morphology and gro:lth rates in an effort to help them improve the quality of their product. I . I . . : The five-year period of this proposed program-project should be wple time to carry i through successfully a number of the projects we are proposing as well.as`to test : tine feasibility of a num'xr of other- L) and evaluate the usefulness of this kind or technology to biomedical science and industry. E. Pacilitics Available " \ Virus Laboratory :, Molecular Biology Department. . Many of the biological c.xperimcnts daescribed here Kll be develo&l; at least 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 facilities of these labora- : tories will be available_, as necessary. c!.Ij:) 12 ' In a+?ition, 8 small, well-equipped machine .' `2'; -. >L?r-' (;;*:::-jr;--J?- .o - . t ~_ ~."- - -- . . . ., . - . e-p - ._ _,__- -. _ .,.-~ , e-2. . ,T'-y--C- T'V, : 7-y - -.. LT i _ - ---,- ._- ---- ;-" - ---.-'-.y.cL- ~ -.I'. mvileged C~unIcation -5 v-e P. - . ..- - -m .-.----- Berkqley &&atory .'A 1 m time to time we.m&y cm, upon q&al shops and:co&& with el&r&s. ' i . the Iwzrence BeY~cley I;rboratorJ to helb us-nith proLLems'r.&.i&`~they may . i. ( : . j.&ave alrcs~ kncvL-= *ibn we can o'"J*z bcILcl in their High 31era~ pPil=j~ics and .rther proms. 'fn addi-. : 6. L._. ..- I f .:wces L.AY o'btain electronic and other sgecL.:,l.ied supplies at very attrzctive and with irraadtite avzLl.abI.Uty from the excellent stockroom facuties of the .. f..laboratoqL We arc very fortunate to be able to take advantn.Ze of ';he sup&b tech- i _. . . . .- 1 !LIhe lar6e+calc automatic equipznt in&ding the computer and flying-spot SCahncr are located in sgecially.remodelled space provfded for that purpose fn the 1 ;i bakcnent 02' the gectrical 3gInecrTn;: &j~.&~, '. ~. t an3 gradu&e studeats Gory E&l.. Members of the faculty cz in-%lectrical'Zngincerin~.ha.ve been talrine an effective role i 1 iAc devclopzent and use of this sgstc~:.. Tnus c&l&or& ill i :-a -. . 1 tiLorl with the ELectrIcal ?&gin- cering Ik~wtnent and the Xn~ineerin~ Research'I;ibor&,o_ri_es offers excellent spcci2.l f f facilities for xorlr of t%e t:,Te tIe.ar& undetiakin~, as well ! for those members of t.hc faculty and. r&duate student: as a unique opportun1~y ' 1 interested in as@yinG their special in Ekctrical Bgi-neerins i . . . skills and !FxlOi!ledge to Sioxedical engineerinS. : L . `. - -Caspus Computer Center - 1 . . . ' . : *_' ' , . , .-:':`ee ' ,.-. ., f . Only node st %nds have beeqbud$ted fc?'use.oZ *he CXQUS Com&Er-Center ;. since, until no:.?, ue have been able to can?J ou$ all the cwuutatlons associated :lith j o*ur work on &r 0Tin co;i;yutinZ system. . .._ When 'ox 0x1 system is satk&sd,' we may te able to reorzanke ou-" yrc~z~s so that some of the pure computation can be-put on I nagnetic t2.p.z and carried out tit the C!ampJs Co:r:puter Center, r:hich offers general facilftiks for large-scale computational work. , ,f'. j-. ,C .a : . . :: _. -. ._ I. . Physics kpartment . - -. Laboratory space in the Fhysics Ikpartrznt is available for this zlork if 1. needed and the excellent rt~ources of the I&chlne Shop and Glansbla?ins Shop can be used frcn time to tic.e as 'necessary. ; . . -3 Jktra-fabrication Space , 1 For fabriCati.cJn cl" xuch bf the sheet a&a? . - _ and t~eZ!ing- I~orI; reqered for .i ! the construction axd I::si:tznance of the large -scale nutqmted zqui.~xentJ l:;'c have been Granted ihe use of a ct-xx~ntnC;I :aztsl building focatcd in t11`- ! . .I I We have had e,rtenzive conversation and in SOEE cases correspoxknc~%ith ail Or the scientific in~zsk$~gators 12~o have proposed projects usfnG ouz equ5pmcn-k Qnd who have vlsitcd our facix-ies. i sincz time, we have not encouraged active the Cyclops has been running only a short i Trork ir our lAooratory until very recently and I . . ,I ~ .. .- - .-..-_w__-... _ _ _ . -_ - _.. _ --__ ._--.. . _ .._ _.-.-. *. `. . . . . ! - . . . ..a ., .:- . . . . 4-2 _ Donald A. Glaser '. * .- ,, y.: )I-4.?:. . . : 'I.,,> : - -, ___ ., ,, : - ,.. __. . .- -_--- - - _ ..__- . . . ..--- __. ._ - _ ., Privi1aCe.d Cormunication - . - - . . . ,. .-t..--.--. .__-- e-.-d -- B---I--~-._"-:-:.---- ---- _. . . . . . , ' `investigators proposing projects listed here harL 7 0 independent support for carrying out these!projects 'in their om l&oratories and TIC intend-to provide use of our. , facilities and nccesscr?, ; supplies ljithout any f&al arraugehehts or exchange of funds- If scheduliw of c:rpcrir?cnts-and as& -ning 09 priorities becozes.difficult, we will ', probably invite so& O- 0 the scientific investigators td join, us and Advisory Cm~ittce to help plan the work schedule. It is too early accurately hog all of these .relationships 3ill develop. so no focal struct*e for collabors'ti.on i, c being planned at this time. .- __- . -. - .- - _-. . constitute an * to. foresee I f i . . 1 administrative i i In addition to thc'scientific investigators n&d above, Professor HerDed B. Baekin ' and Professor Martin Grahun of the D..pmtment of Electrical Engineering and Computer : Science, Un1versi.ty of California, Berkeley have been very helpful in giving advice j concerning'con:puter hardijare and eoftmre. !Thcy generously agreed to continue . ; this relationship and perhaps plsy a nom activ -e role in this prograrr in comir?g years. j . . , I G. Principal Investigator Assurance. 1 , , - I The undmsigned agree, 4 to accept responsibility for,the scientific and' i technical conduct o? the research project and for provision of required progress reports if a grant is wmrded as the result of this application ' .I 1 \ . 9 Novenbcr 1973 Date . .- Principal Investigator _ : I . * . . . .