- 26 Glaser, Donald A. Privileged Communication , fs iii. Ordering of mutants within the close neighborhood of Bich ‘other can be done by two and three factor crosses by generalized transduction and also by a new episome complementation method developed and described by Robert N. Reeves and John R. Roth, JMB 56, 523 (1971). Use of automatic techniques will allow the enormous labor required to make an intensive map to be done easily using transduction, mating, and other techniques that can be carried out on agar. The establishment of a large library of temperature sensitive and more completely characterized mutants covering the chromosome map thoroughly would have very many applications in the study of bacteria and especially of yeasts and higher organisms. We propose to begin the work with bacteria for which the techniques seem straightforward and extend it later to higher organisms. Significance: i. By periodic measurement of map distances by cotransduction or interrupted mating one can monitor increases and reductions of the chromosome by the net effect of gene doubling, recombination, deletion and other processes that may affect its size. With a large number of standard markers and standard proce- dures the machine can keep a steady picture of the state of integration or autonomy of various plasmids, of the chromosome number, if that is subject to change, and of the size of the chromosome... It seems more. likely that the size of the chromosome is not an accurately conserved quantity but there will be variations in the population and it is hoped that methods of measurement will be sensitive cnough to make some description of this distribution and how it changes when the parent strain of the population contains various mutations especially affecting DA replication and repair. , ii. There may be regions of the map for which no temperature sensitive mutants or other conditicnally lethals can be found., It is of great interest to know how much of the DNA specifies no function and is functionless except for its role in evolution of new genes to carry out new functions or for structural functions at the DNA or RNA level. iii. When the whole map or at least regions of it are densely filled with markers it may be possible to discern overall patterns of placement and organi- zation of the genes according to their function or evolutionary history and thus to understand better evolutionary or physiological demands that led to this particular pattern or structure. The operon concept is the most obviously important fact of this type but there may be peties as yet unrecognized. iv. It will be possible toa supply large numbers of densely located temperature sensitive anu other kinds of mutants in particular regions of the map for inten- sive further study of particular problems in this and other laboratories. We intend to use the method immediately for trying to generate large numbers of mutants in tne neighborhood of known sites for DNA regulatory mutations hoping to discover other DNA regulatory mutants in the same neighborhood. As the techniques develop we will probably be able to supply other laboratories with large nunbers of mutants important to their particular interests. If eutomation makes it possible to follow changes in a densely-mapped bacterial chromosome subjected to a variety of mutational situations, the resul- ting insipht: into chrenosene mechantes will be extremely va tuapics axtension Gv Ghis appreneh to ehiuccosa: ml dpbrner i her irmortene conecaucnecs Vor witereiandine a vide rane oe eilitdes pacvunetoaac gor cecil in which * good biological mechanisms of genctic recombination are available. _ 27 « Glaser, Donald A. Privileged Cormunication * , 4) Genetic characterization of the chromosome terminus and the regulation of cell division in E. coli. , David R. Zisman, Assistant Professor of Bacteriology, University of California, Berkeley. Ss Methods: Recent evidence from gene frequency measurements (1-3) autoradiography (4) and biochemical analysis (5) demonstrate bidirectional chromosome replication in E. coli. The origin of replication appears to map at about 75 map min while the terminus has been mapped at about 30 min (6). The termination of chromosome replication appears to be necessary for chromosome segregation and subsequent septum cross wall formation (7-9). - . It has been suggested that chromosome termination may trigger division by the - transcription of division related genes, located at the chromosome terminus, at the time of their replication.(7,10-15). This hypothesis has recently received some experimental support: (a) studies of cell division following DNA, RNA, and protein inhibition at the time of chromosome termination in synchronous cultures (16-18) in@icate that the specific replication of the lest 0.5% of the chromosome. (0.45 map min) is required for subsequent cell division; blocking protein synthesis during this replication will block the subsequent cell division. (b) Several filament forming septation mutants have been obtained which map near 30 min, the chromosome terminus (15, 19-20). : Unfortunately, the region of the genetic map around 30 min is one of the most poorly understood areas (21). Very few markers have been identified; a stable F' has never been isolated for this region (22). We therefore propose to study this region of the E. coli map in great detail using the automated techniques now available. Hopefully the study of this region will help us understand the nature of the link between chromosome termination and cell division. We have isolated a man~ mutant (30.5 map units) that is non-reverting. We propose to use the transducing phage P, to cotransduce mutagenized markers (23) from a mant strain to our man~ strain. Transductants grown on mannose minimal medium will be plated out using Dr. Glaser's automation equipment, replica plated at different temperatures, and temperature sensitive colonies obtained. These colonies will be characterized for nutritional defects or division defects. The nutritional mutants will be saved to help us map this region of the chromosome. The division-membrane mutants will be studied more carefully to determine possible relationships with chromosome structure and/or regulation of division. Complementation studies should indicate the specific number of division related genes localized in this region of the chromosome and the possible existence of a division operon. Double mutants will be prepared so that the in vivo interaction (epistasis) of known mutants of different phenotypes can be studied (15). This approach can lead to the sequencing of related gene functions and is the first step necessary to determine the ordered pathway for septation in a manner similar to the study of T-even phage development and other self assembly systems (2). i. Masters, M., Proc. Nat. Acad. Sed. U. S., 65, 601 (1970). 2 ‘ ‘ey aves £5c, 1b? (1971). ? ? o 2. Bird, 2. B., Lovern, J., Martuscelli, d., and Caro, L. G., J. Moi. Biol., 70, 549 (ly72). | Mey ek says . eye ue aa Se ROSverS, He, Od Loode, F. 28 - Denald A. Glaser Privileged Communication a! 20. 2c. 23. 24. —§) Prescott, D. M., and Kuempel, P. L., Proc. Nat. Acad. Sci. U. S., 69, 2482 (1972). McKenna, W. G., and Masters, M., Nature few Biolozy 240, 536 (1972). Hohlfeld, R., and Vielmetter, W., Nature Tew Blolozy 242, 130 (1973). Clark, D. J-, Cold Spr. Harb. Symp. Quant. Biol., 35, 823 (1968). Helmstetter, C. #., and Pierucci, 0., J. Bacteriol., 95, 1627 (1968). Walker, J. R., and Pardee, A. B., J. Bactcriol., 95, 125 (1968). Clark, D. J., J. Bacteriol., 96, 121% (IS68)._ Hirota, Y. A., Jacob, F., Ryter, A., Buttin, G., and Nakai, T., J. Mol. Bioi., 35, 175 (1968). | : Pierucci, 0., and Helmstetter, C. E., Fed. Proc., 28; 1755 (1969). Previc, E., and Richardson, S., J. Bacteriol., 97, 416 (1969). Pritchard, R. H., Barth, P. T., and Collins, J., Symp. Soc. Gen. Microbiol., 19, 263 (1969). ' ' Zusman, D. R., Inouye, M., and Pardee, A. B., J. Mol. Biol., 69, 119 (1972). Jones, N. C., and Donachie, W. D., Nature New Biolozy, 245, 100 (1973). Dix, D. E., and Helmstetter, C. E., J. Bacteriol., 115, 786 (1973). Marunouchi, T., and Messer, W., J. Mol. Biol., 78, 211 (1973). Hirota, Y., Ricard, M., and Shapiro, B. In L. A. Manson (ed.), Biomembranes, Vol. 2, Plenum Publishing Co., New Yors, pp. 13-31 (1971). Ricard, M., and Hirota, Y., J. Bacteriol., 116, 314 (1973). Taylor, A. L., and Trotter, C. D., Bacteriol. Rev., 36, 504 (1972). Low, K. B., Bacteriol. Rev., 36, SO7 (1972). Hong, J., Smith, G., and Ames, B. N., Proc. Nat. Acad. Sei. U. S. 68, 2258 (1971). Eiserling, F. A., and Dickson, R. C., Ann. Rev. Biochem., 41, 467 (1972). Significance: Detailed understanding of the relationship between DNA synthesis and cell division in E. coli may give important insights into the same relation- ship for proliferating animal cclls, which generally do-not synthesize DNA except in preparation for cell division. som Studies in biochemical evolution in E. coli and B. subtilis. Joshua Lederberg, Professor of Genetics anc Biology and Cnairman of the Genetics Department, School of Medicine, Stanford University, Stanford, California. We wish to observe alterations in polypeptide products resulting from mutations in synthetic genes (generally synthetic horopolymer sequences) which have been inserted into the genomes of E. coli and B. subtilis bacteria. Immunochemical methods will be used for detecting these alterations by examining large numbers of small colonies for which no biological selection condition is known. By observing evolution of a polypeptide, much can be learned about the genetic code and about rates of various kinds of mutations in different nucleotide environments. Genetic organization of the E. coli chromosome: mutation rate versus map position of the translocated lactose operon. Gordon Edlin, Associate Professor of Genetics, University of California, Davis, California. The purpose of these experiments is to probe the genetic organization of the FE. coli chromosome. Ultimately we would like to understand why genes are located at particular sites in the chromosome. One approach to this question is to measure frequency of mutations in a gene (or genes) which have been translocated co nudr of divverony sives in the ehvemusen:. A model syste. for theee ex- periments is provided br the luesase oneron. A set of stroins exist vhich are genetically unifora except that the lactose genes have been translocated to a Privileged Communication . — Wii Ce 7) ~29- Donald A. Glaser number of different sites in the chromosome. These strains will be mutagenized with a varicty of mutagens (nitrosoguanidine, ethylmethane sulfonate, U.V. light, etc.) and the frequency of lac’ — lac” cells will be measured. Preliminary studies have shown that the frequency of mutations in the lactose genes are a function of chromosomal location. After analysis of the lactose genes, the same analysis can be applied to other genetic systems such as an amino acid biosynthetic pathyvay, ribosomal protein, etc. Genetic techniques for constructing the appropriate bacterial strains already exist. WeEnclosed is.a brief statement for your grant. We would like to go ahead on this as soon as possible since it is all worked out and is basic- ally a matter of cranking out the. data. The diagram shows the nine strains we want to test. The lac genes are located at the 9 positions wv hee 7 ne we an ee e . We will mutagenize with ENS wand nitvosoguantdine for starters. We can measure the mutagenesis here by measuring the number of valine resistant colonies. That gives us a number to use to normalize the mutagenic effect- iveness. We would then bring down the mutagenized culture to be sprayed onto trays. We want to test the number of lac” cells. I think the easiest way to do this is to place them on EMB lactose agar. Lac? are red and Lac” are white. We probably need to photograph at 2 or 3 times to reliably distinguish the 2 types and probably have to set some limits in the computer as to what it calls white and what it calls red so we probably need a dry run. Once that is determined we can run them as fast as time allows. I presume we will work with Phil on this. Let me know how and when you want to proceed. “ Recombination deficient mutants of E. coli. . moe Alvin J. Clark, Professor of Molecular Biology and Bacteriology and Immunology, University of California, Berkeley. Method: "Our worl in large measure stems from the discovery of recombination deficient mutants of various recombination preficient strains of E. coli. In doing the necessary mutant hunts the present bottle-neck is the picking of colonies of survivors of mutagenic treatment and patching them in geometric array. I am very interested in testing the dripper you have invented as a means of depositing cells in ogonetric arrey prior to testing their clones for recombina- tion deficieney. Is te very noocible this isc feellitete meny Capea net s we have 2 ° 2 n 3 been sitting on because of the Lasor involved in piching and patchinc. 9) 10) 1) Donald A. Glaser Privileged Communication gy i ° ~ New Salmonclla typhinuriua tester strains for cetecting mutagens and carcinogens among environmental chemicals. Bruce Ames, Professor of Biochemistry, University of California, Berkeley Method: This work is an extension of work already published to special cases for which the labor of mutant isolation and characterization is Limiting. i. ‘Ames, N. B., Lee, F. D., and Durston, W. E., Proc. Nat. Acad. Sci. U. S., 70, 782 (1975). 2. Ames, N. B., Durston,W. E., Yamasaki, E., and Lee, F. D., Proc. hat. Acad. Sei., 70, (2281 (1973). Fine structure mapping in the histidine operon. Bruce Ames. Method: This work is an extension of work already published to special cases for which the labor of mutant isolation and characterization is limiting. 1. Ames, N. B., Lee, F. D., and DUEStOn, W.-E., Proc. Nat. Acad. Sci. U. 5., 70, 782 (1973). 2. Ames, N. B., Durston, W. B., Yamasaki, E., and Lee, F. D., Proc. Nat. Acad. Sic., 70, 2281 (1975). Metal ion mutagenesis and plasmid curing in Salmonclle tychimurium. Peter Flessel, Assistant Professor of Biology, University or San Francisco, Sean Francisco, California ? Method: "I have been looking at the interactions of metals with bacteria using tuo assay systems. First, I have been studying metal ion mutagenesis and second, plasmid curing by metal salts. The decision to focus on metals was based on the ‘near presence of a colleague in the chemistry department who had been studying metal carcinogenesis for fifteen years anl was eager for some company. "The work to date has been basically an extension of Bruce Ames' scheme applied to metals. So far I have shown that MnCl and NiCls are mutagens in S. tymphi- murium. Our search for new metal mutagens is contiauing and I suspect we will find others in the next tev months. The mechanism of metal mutarenesis has not been thoroughly explored. It is not known, for example, whether metal ion pene-~ tration of the cell membrane is a prerequisite to mutagenesis. To find out, I would like to select for mutants which are temperature sensitive for resistance to metals. The assumption is that resistance would be a reflection of the fail- ure to take up the metal.. I would select for growth at 42 (permease denatured) and no growth at 37 (permease functional) in the presence of the metal. Having obtained such mutants I would test them for susceptibility to metal mutagenesis at both temperatures. I realize my proposal is perfectly straightforward. If I carry it out. with the time and resources at my disposal, it is at least a year's work. ith the "“dumbwaiter" I think I could have the first mutants in several weeks. ft. . tt ae t + - e = < . , - 7 _ Pd + C - . 4 Woes en virulence in Calmonolle and isolation of mutants suitable for «a 34 . enn eee eee coe ave vVacoun Bruce Stocker, M.D., Professor and Acting Chairman, Department of Medica wm ey : 2 ie ~ sytes Donald A. Glaser _._erivileged Communication oo. i? — Microbiology, School of Medicine, Stanford University, Stanrord, California. For experiments on the mapping of genes affecting the virulence of Salmonclla. species, it is expedient to obtain genctically marked sublines in particular strains. For example, in lines of S. typhimurium which differ-from the available genetically marked stocks of strain LT2 by their high virulence for the mouse, - on intraperitoncal inoculation. It has been the experience of several. workers that: auxotrophic mutants obtained by mutagen treatment of virulent strains of | Salmonella commonly nave unvanted additional mutations causing reduced virulence, by unknova mechanisms. Therefore, in theory, the best method of procedure would be to introduce choren negative alleles, determining nutritionel requirements. or inability vo ferment particular substrates, by transduction. To do this by {ordinarily available methods, even with the aid of penicillin enrichment, is hardly practicable, because of the amount of labor required to detect the rare transductants, which cannot be selected for. Dr. Glaser's apparatus should make possible the detection and isolation of the desired transductants by an automated st procedure. temperatures. a . 14) Isolate mutants of Bacillus subtilis resistant to certain phage and to drugs a like p. hydroxyphenylezouracil for studies on DNA synthesis. . A. T. Gonesan, Professor of Genetics, Stanford University, School of Medicine, : CO Pee t) BM at Seantord, California. : Our research project involves the study of the mechanism of DNA replication and ‘ ty 15) - oe ryt me S06 ne i we cyt ca ne ce ee nee eee oa eee fener ee meee em ot Privileged Compynication Coaqynwiien set oe 22 - . Donald A. Glaser.......---. Lo eee eee its genetic control in Eacillus subtilis, a transformable bacteria. We have isolated several temperature scncitive mutants that are defective in DNA synthesis The thermoscnsitive protein has been studied in a few cases. There are about 9 groups of genes that control DNA synthesis. There may be even more. We isolate ‘these mutants routinely by conventional, slow and laborious procedures. The automated petri dish machine would be ideal for the above project. We are speci- fically interested in both low and high temperature sensitive mutants, and mutants that are resistant to drunps like p. hydroxyphenylazouracil. This drug specifi-~ cally inhibits DiA polymerase III in Bacillus subtilis. Polymerase JII is directl involved in DNA synthesis. Resistant mutants vould help to locate the position ~ of the gene for the enzyme. The system is also adaptable to test phage mutants which are currently studied. The instrument is a very valuable and unique tool for our projects. We would very much like to collaborate with Dr. Glaser in obtaining several important mutants and adapting the machine for other related projects in cell biology. Sercening for possibl @ mutagens among environmental chemicals by mutations affecting sporulation in Bacillus subtilis. Lawrence E. Sacks, Research Microbiologiss and James T. MacGregor, Research Pharmecologist United States Department of Agriculture, Agricultural Research Service, Western Regional Laboratory, Berkeley, California. ‘ , Thousands of chemicals, whose biological effects are little understood, have been disseminated into our environment and the food we eat by modern technological. society. Most frightening of these chemicals are the mutagens, with their poten- tial for teratogenic effects, cancer, and unknown long-termn effects of alteration of germ-celle. : In screening for possible mut tagenic chewieals, microorgenism systems offer the ; advantages of speed, simplicity, and economy over animal systems. A disadvantage of microbial systems now in use is that they test only for mutations occurring in one or a few genes. A bacterial system sensitive to mutations on many. genes, scattered throughout the chromosome, would seem to offer important advantages over currently used systems (1). We believe such a system is that governing sporu- lation in the genus Bacillus. Sporulation is a very complex process requiring the participation of a minimun of 28 operons for the sporulation process alone(2). Other systems (e.g. TCA cycle) are required for successful sporulation. Eight ~ hundred genes have been estimated to be required for successful sporulation (5). Selection of asporogenic mutants is simplified by their characteristic white color easily distinguished from the wild-type brown colonies, colored by formation of a pigment late in the sporulation of B. subtilis,Marburg strain. Using a highly transformable strain of this organism, and a wide variety of mutagenic agents, many sporulation genes have been mapped (4) in programs designed to unravel the genetic control of sporulation. We propose only to invert this procedure, and to use sporulation mutants to identify new mutagenic agents. . Dr. Glaser's instrunent, capable of identifying single mutants in huge populations. will be of great value in identifying mutagenic activity at very low concentra- tion levels. This combined use of a bacterial system involving over a hundred genes with scanning by an instrunent cavable of identifyinz mutation rates below ' 1077 chords 3 “s4 QV crouse mrvbte tees dsr one meotssive rvos fax tdent aryt nS we wots g MD ae ee SD ey = muturenie choideals. PAs she ULaotl Privileged Cormunication =! We summarize below some advantases of the proposed. system: : t ~e , ‘Ll. It is based on forvard mutation, the mest general type of detection eyateri. - Any type of mutation which inactiviates or substantially alters a gene essen- i tial for sporulation will be detected. 2. A large number of genes are involved in sporulation (2, 3). Some mutagens ae are specific for particular regions of the DiA.- The more genes surveyed, the . .- less chance of excluding mutagenic “hot- “spots” . N 3. Sporulation mutents are often characterized by a block at 2 ‘particular stage in their morphological develomment. The frequency of occurrence of particular stages of arrest vill permit an assessment of the randomness (or specificity) of each mutagen. . 4, The Be. su subtilis system is well-suited to genetic studies. Highly transrorna- ble strains exist, and many genes have alreedy been mapped (4). Dr. Gleser's . scanning system, hovever, is not limited vo the pismented B. subtilis colo- nies. Other well-studied species (e.g. B. meraterium, B. cereus) may also be Lo employed. fe SS References ; ‘ 2 \ . tt i 1. Hollaender, A. (Hd.) (1971). Chemical Mutazens. Princinles and Methods for i i Their Detection. Plenum Fress g, iN Y. Vol. I, TL, III (1973). i 6% ! : . 4 Le ; wa I 2. Pigeot, P. J. (1975). "Yemnin ng of asporozenous mutations of Bacillus a j suotilic: A minimum estimate of the number of, sporulation operons”, " J. : ae Bacteriol. 114:12h1-53. Pe , ue 3. Balassa, G. (1971). "The genetic control of spore formation in Bacilli", ' Current Topics in Microbiolory and TmunoLocy 56:100-52 } : 1 4. Young, F. E. and Wilson, G. A. (19/2). "Genetics of Bacillus subtilis and other gram-positive sporulating Bacilli." in Spores V (H. 0. Helvorsen, R. Hanson, L. L. Campbell, Bis.) pp. 77-106. 3 me 16) Proline degradation mutants in yeast. ee John k. Roth, Associate Professor of Molecular Biology, University of California, . Berkeley. . We've started looking at proline degradation in yeast i ‘ny: . - Here the available mutant 4 : . —_— : men | ——.earichuent technicues, generally wor: poorly. Most pecple seek yeast mutants in a. | fairly “brutc-force" sort | of vay. A large set of proline-non-utilizing mutants would be very useful to us. This hunt would need to follow the Salmonella hunt and probably should follow preliminary work (in progress) on the few available ! mutants. In this way the most advantageous conditions can be determined. , : - i . . ; i :17) Saturation mapping of one yeast chromosome. . , ~ i John R. Roth Yeast has roughly 4-5 times as much DHA as bact eria- Roughly one hundred genes ' have been located. The snecine of these sence are wide enough to make it diffi- ia oe . ; . ne _. cul Soueh nove. Ag Senon (ey cia oo VoUY aubecaturetiion cc) WSR Pate Gh UR ON eh Wee OTP ED wal ty wVOULL give new moviora tor mapsing anti eo minire:: ertimeste of “tne mene density. te ae ce cp apnea ee OS EN ee ne faeee oe etme armament #05 20) A hunt for temperature-sensitive mutants should yield mutants carrying lesions in & ; ' Qcveloprent of genetic mass Donald A. Gloser Pantin tye mae ~ 3h - : . c. vie aes wo Tree Privileged Communication .oo i _=_™» _ we cessed cess Strains will be obtaincd through Robert Mortimer which are monosomic for a small chromosom?. Other strains will be: obtained which are monosomic for a large but ‘poorly marked chromosome. These strains are diploid for all but one chrouosome. the chromosome for which these strains are haploid. These can then be analysed. Determining the number of genes involves is fairly easy because of the simplicity of yeast complementation tests. I'd like to try this in a year or so after I've gotten back from sabbatical leave. (I'L1 be doing yeast genetics during that, tine.) . . Genetic mapping in Saccharomyces cerevisiae Robert K. Mortimer, Proiessor and Cnairman of Medical Physics Department, Univer- - sity of California, Berkeley. — oe re The availability of detailed genetic maps is an important component in determining the suitability of an organism for genetic and molecular studies. For a number of years, we have carried on a program of cenctic ma os in the yeast Saccharo- myces cerevisiae as an adjunct to our other studies. ese mapping studies have | resulted in a genetic map which establishes the lo cation of more than 150 genes on 17 chromosomes. However, because of the large number of chromosomes and the hign 7 frequency of genetic recombination in yeast, very few heavily mapped regions are | availavle. Such regions are important for studies ve wish to carry out on gene conversion and its relation to mechanisms of genetic recombination. We believe | the instrument developed by Professor Glaser could help to speec up the further in this crganisn. The avproach ve propose to use is based on the random snore te ecanigue deserived in our recent mapping paper (Morti- mer and Hawthorne, Genctics Te 2 55-54 (1973). A cerics of strains ¢ that each carry, one of a set of cchre sunnressors in combination wi ha 2 suppressible canavanine resistance gene and an additional selection of nutritional genes vill be crossed to a large series of tenperature sensitive lethals The resultant crosses will be sporulated, and the asci will then be treated with glusulase followed by sonicea- tion. The sonicated suspension vill be inoculated onto complete medium containing canavanine. Only spores lacking the suppressor and carrying the resistance gene will grow. These can then be transferred by replica plating to a series of "arop. out" plates to score the nutritional genes and toa W360" environment to score the conditional genes. The patterns of growth: nongrowth on these various | media can then be recordcd automatically by the scanner and the resultant data~* } analyzed for linkage by a suitable computer program. In this procedure it will te necessary to inoculate at a concentration that reduces to a low level the proba- bility of clones developing fron more than a single spore. The instrument should greatly facilitate random spore analysis both by permitting larger samples to be analyzed and by automatically recording and analyzing the results. ae ct t Gene conversion and recombination in unselected mitotic yeast cells. | eymour Fogel, Professor and Chairman of Genetics Department, University of California, Berkeley. Post-niclotic segregation and heteroduplex. DNA. . Seymour Fogel. f Tio rather specific pronosnls for use of the atometcd mierobioloyi.cal. equiprent are Peocented. Ghose aS tabhwes health rele: Ts, Che PoOaGe Se caepren sito: el LO: dn unseloeted Gerba. ecaregation in yeast conld provide a model system, a senvia Wy: adinsset o 47 4 ne Lat ea nas wea Tes radar wat yen wenn - we ae “3 te Feg yt tae ae ‘Our currcnt understand awa Tht . son pant ye : a ey prec ete cee POLE LVS CO t abe Cui > ' i sihpeueiy xy te ifs ~ 35 - - Donald. A. Glaser Privileged Commmicction SD strategies and overall rationale may be carried over to address such seemingly unrelatcd though central problems as chromosome nondisjunction,, or screening mutagens, carcinogens, fungicides and antibiotics for their genetic effects. I. Gene conversion and recombination i unselected mitotic yeast cells ing of intragenic recombination in cells committed to a mitotic cycle emerges fron data generated by selective methods. In effect, these: depend on appropriate signal devices that lead to the detection and recovery-of Only wild type or prototrophic recombinants. However, we have recently’ demon~ strated the occurrence of mitotic co-conversion in hybrids marked by three of four heterozygous sites in a single structural gene, and it must be emphasized that multisite conversions do not typically generate wild type recombinants. Thus, though co-conversions might represent the most frequent event class, they remain uncetected and unscored in conventional selective procedures. By. analogy to our studies on unselected couplete meiotic tetrads, we propose to analyze (in the same hybrids) an unselected vooulation of mitotic cells for all econversional events falling within a defincd genetic region. . Mitptic gene conversion in yeast occurs with an everase frequency of the order 10“" to 107°. Accordingly, collecting a sample of 10° or 10° unselected conver- . sional events involves sercening a total vepulation of 10” - 10% cells, ora sample beycnd the capability of routine microbiological methods. Automated ; microbiolocy equipment, however, augurs well for the successful completion of this and cimilar studies. , ¢ a) automated single cell innocula; b) replica- c) irradiating the replica prints (UV or X-ray); rieving sectored clones; e) finally, complete 5 ored clone by random spore or tetrad analysis of Our analytical setratezy reouire plating the derivative clones d) detecting, locating, and genetic diagnosis of each ¢ each sepment. Tr . ’ Bs e + & zr ec ‘ mee eee II. Post-meiotic sesrecsation and- heteroduplex DNA. Common to all molecular models seeking to account for genetic recombination are enzymatically mediated steps at eventuate in heteroduplex or hybrid DNA produc- tion. At the in vivo genetic level, the presence of unresolved heteroduplex DivA is detected by cost-meiotic segregation (FMS). PMS is comparatively frequent ~ among the total aberrant octads of Ascobolus or Sorderia. However, technical difficulties with these forms, including a paucity of genetic markers, preclude: total and critical analysis. With autorated microbiological procedures adapted to random spore or tetrad analysis based on diploid yeasts suitably marked with ‘(-lO0 heterozygous sites (i.e., loci and alleles of Imovwn meiotic conversion frequencies), we could readily assess the frequency, extent and distribution of heteroduplex DNA in the yeast genome on a statistically reliable base. Sectored ascosporzl clones, otheruice concordant for all segresating marzers will be considered as PMS events. a Lo . — Also, from the distribution of BitS events among spores produced by heteroallelic diploids of the type ++/12 or 1+/+2 (notation as before) where the mutant allele | pairs may be chosen from extensive fine structure maps to represent a range of i genetic distances, the resularities and bicie attributes of heteroduplex DNA . Oo nr Sy aad. a > Lat Oho Dia MTG er COS ue LpYerroou, tee awe 7 none weer ee eee ates Fee nee ete eee et eee tet ee ene ad ee 7 Ne eee: ees NT Se Ran tN es ame oe a ’ t : te a ee waetis Hepa eee 8 35a _ . - Donald A.. Glaser. ne Frank Ruddle, Professor of Biology and Human Genetics, Department of Biology, - Yale University, New Haven, Connecticut. FS I believe that your machine has particular possibilities with regard to the recovery of conditional temperature sensitive mutants in tissue culture populations. As we have previously discussed, it would seem possible to esta- blish colonies in the machine and then to shift to higher temperature and examine the colonies for retardation in the rate of increase of colony size. It would be possible to maintain the cultures at 34°C as a permissive condi- tion and then to increase the temperature to 38.5° for 3 hr. periods out of a total period of 24 hrs. and carry this regimen forward for a period of one or two weeks. It would seem to me that this would not kill the temperature sensitive mutants but.would result “in a decided difference in their colony size which could be easily monitored by your photographic equipment. The iso- lation and characterization of temperature sensitive mutants will, I believe, be one of the most important aspects of somatic cell genetic work in the next decade. It should be possible by this means to obtain mutants which affect the biosynthesis of cell membranes, nucleic acid, and protein. It is also possible to:pick up mutants which specifically affect the ability of mammalian cells to progress orderly through the cell cycle. All of these mutants can be analyzed by genetic complementation tests involving cell hybridization and Chromosome segregation. For this purpose it would be best to make use of Chinese hamster cells or mouse cells as the population in which the mutants are recovered, , It seemed to me that your machine could be adapted also for recovery of . mutants in differentiated cells. - Quite a number of tissue culture cell lines which express specific differentiated traits are now available. For example, we are growing hepatoma cell lines which produce albumin. ‘The albumin is secreted into the medium at high levels. -It would seem to be possible to maintain colonies and then test the individual colonies for albumin production perhaps using a fluorescent reagent. One can then examine a large number of colonies for cells which fail to produce albumin. This would represent -an excellent method for picking up non-producers. These cannot at the present time be enriched by selection techniques. One could also test for reversion to capacity to produce the differentiated product using the non-producing mutant as the base population. This kind of procedure could be adapted to cell © lines which produce hemoglobin, myocin, nerve specific protein, etc. ’ When your machine is sufficiently developed to make use of mammalian cell populations, I would very much like to be in touch with you with regard to these possibilities. If you are interested in pursuing these possibilities I'd be more than happy to come out to Berkeley and spend a month or so‘in this connection. By Privileged Communication =: — cette 20a) Mammalian somatic cell genetics. : Dee ee eee mee ee reper pam ee ae Corie oetian en 26. . Donald Ae Glaser. PDs pep - 7 O : ~ 7 Nw = Privileged Communication, — eee ey Ts solate and characterize a ‘larg a number or -steroid- and cyclic AuP-resistant ‘clones of mouse: lymphoma cells.: Gordon M. Tomkins, Professor of Biochemistry, Uni versity of California, San Francisco. : ; ' ae wane ne |. General Oojectives: For some years our laboratory has been studying biological ~~~ = regulatory mechanisms in cultured mammalian cells. We have concentrated primarily | on the action of the steroid hormones but more recently have become interested in the’ cyclic nucleotides as well. The bulk of our work heretofor has been a bio- : | chemical analysis of the molecular mechanisms of ecll-hormone interaction. Quite i recently, hovever, we have begun to explore genetic technicgues to pursue our ob- | : Jectives. For this purpose we have been using cultured mouse lympharn cells which: i are killed on prolonged. exposure to ‘either the adrenal Glucocorticoids or to cyclic AMP. Thic response occurs at vhysidological levéls of the effector’ molecules and i presumably reflects the well known irmunosupressive action of the glucocorticoids : and of agents which elicit cyclic nucleotide synthesis. In any event, we have beer. { able to sclect variant lymphoma eclls resistant to the killing actions of the steroids, cyclic AIP or both agents. Our results to date indicate that the transi- | tions from effector-sensitive to effector-resistant occur at random at a rate, i in.the case of the steroids, of 3 x 10-° per cell per generation and for cyclic i AMP, of approximately 1 x low? per cell per generation. Various mutagens increase the frequency of steroid resistant cells. Biochemical analysis of the phenotypes | of steroid- and cyclic AMP-resistance had indicated that in the former case, three types of variants can be isolated: those lacking the normal cytoplasmic steroid 1, _ binding activity; those where binding takes place, but in which the receptor- |) steroid complex is not translocated to the nucleus; ani finally those in which binding and translocation occur but céll death does not result. Preliminary investigations suggest that various phenotypes also give rise to ; cyclic AMP resistance. To date we have studied only cells in which the cyclic nucleotide binding protein and its associated kinase are deficient. | Specific Aims mo I.” To isolate a large number of steroid- and cyclic AMP-resistant clones of lymphoma cells. @. To determine the frequency of their occurrence and the effects of a variety of natural and artificial mutagens on the Uransi Grou from sensitivity to : resistance. . 2G 3. To determine the biochemical bases of cell killing. ‘ 4. To characterize the: PREnot types: in terms of various known steps in hormone action. - : 2 To carry out complementation analyses using cell hyoridization techniques to determine the number of piochemical steps involved in cell-hormone interaction. oo, . . 3 6. To determine whether the transitions result tron menetic or other types of i i - stochastic, heritable variations such, for example, as might occur during ‘ i . the differentietion process. ; : ; ' [. To investigate possible relationships between resistance to the steroids and | i to the cyclic nucleotides. -%. To apply similar methods to circuleting malignant cells in patients with { } * lymphoma or leukemia in an attempt to design more. rational therapies for : these discases. aoeniteonunes: Uhe projected sbecics bear on men sepects of cell IstoLory ant ee crm Reet eee 23) From a theoretical point of view, these experiments could provide novel approaches) Ceytinuntion ogee 3ST = - Donald A. Glaser... eee. Oe Febiilegsd comunicetion sD ene ae clinical medicine. The glucocorticoids are ma jor ‘therapeutic agents in ‘Leukemia’ ‘and in other malignancies. Their etfectiveness is limited only by the emergence of horuone-resistant cell populations. Our observations With cultured cells can’ ‘therefore serve as a useful rodel for studying how it might be averted. The finding that certain mutagens, in particular alkylating agents, enhance the con- version from steroid-sensitivity to steroid-resistance already indicates that ‘therepeutic regimes which employ alkylating agents together with steroids might be. redesigned to avoid the possibility that steroid-resistan t cells are produced in . the course of therapy. . v i ' These studies also suggest that new classes of agents, such as the cyclic nucleo-; tides or compounds which elicit their production, might be used in tumor chemo- therapy. The apparently lower frequency of resistance to cyclic nucleotides holds out the hope that these agents could be more effective therapeutically than ‘the steroids. . . So ‘ to investigations of drug and hormone action by combining geneties, with cell biology and biochemistry. It should, for example, be possible to isolate cyclic AMP-resistant: variants in which adenyl cyclase or various specific membrane recep- tors are deleted making it possible to study the interrelation between the elements in this important regulatory circuit. The same considerations hold true for the steroid hormones and studies on their mechanism of action. Steroic and cyclic AMP-resistance are the result of changes in structure of the recepuors. Since these molecules have been identified, and to some extent, puri- | fied, the generation of resistant mutants can be correlated vith altered molecules: Therefore a more complete genetic analysis can be carried out than if the selec-~ tive 1arker (e.g. drug resistence) were not correlated with a known protein. Linkege analysis in marmals by somatic cell genetics. Theodore T. Puck, Director, Institute for Cancer Recearch; Professor of Biophysics: and Genetics, Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center, Denver, Colorado. Preliminary discussion of this project has indicated the great labor of isolating mutants and establishing linkage. Feasibility studies need to be carried out . before real research plans can be made. Because the genetic exchange system is ! so inconvenient compared with E. coli, the automation may be even more. valuable for animal cells than for bacteria. Sensitive detection of muta agenesis by changes in colony morphology-extension to. additional bacterial and eukeryotic cells. : D. A. Glaser. : i Method: Since colony morphology is a highly polygenic characteristic, it should be a very sensitive detector of mutagenesis. Extremely uniform reproducible culture conditions are peauines to guarantee reproducible colony morpholozy even~ in the absence of mutations. For measuring gross mutagenic effects down to very low "exposures", we plan to explore the limits of colony reproducibility for a variety of organisms. ; ; ‘ succesrtul, colony morpholosy chantes provide a mechod of copie ePfucte on a wide variescy of clornable cells, even if Litile BEE . 38 _ Donald A. Glaser. . Privileged Communication >. ple sce a neers we tar mmeen caren coe thin sega eiennees Satie amano Hosa See ne ee ee ; or no genctic information is availeble. Screening of chemical and physical i mutagens is an “obvious application. . ; i : ' wo ee eee, 24) Transformation and mutation of Mamraiian Cells in vitro by low dosés of matagens and ionizing radiation. D. A. Glaser . s Oe ee rete mennntennter eon ang-E. J. Hall, Neture 25, 450-455 (1975). Exbryos of golden hamsters were ¥y minced and separated into individual cells groving on agar. The cells were irradiated with 1 to 600 rads of X-rays, incubated, stained, and the colonies forned 1 (probably about 2 uu in size) examined for forms made by transformed cells . , 5 j bo , “Transformation of Mermalian Cells in vitro by Low Doses of X-rays", C. Borek i ' t i j AO + eee meee Table of Effect of Transformed Cells ‘Dose — oe Clones Examined Cells Transformed 0 - %6,000 Oo | PS Po = 7,900 3 | , 10 , 10, 200 4 i - 25 oo 5,500 8 | Clearly large numbers of clones were examined for the infrequent event. The ability to use larger nuzbers of cells and examine the clones formed from them would make the numbers found more precise and allow better description of the dose response is curve at low doses 25) Behavioral Mutants of Motile Organisms —— D. A. Glaser ; ~ . . .- at mM. In the original provosal for construction of the DW and scanner system, we de-. seribed possible behavioral studies of motile organicms of standard or "instinc- tive" behavior as vell as adaptive or "learned" behavior. ‘The following is-quoted as an example of the type of study we would like to pursue sometime during the next few years. i ke ee ee ne ee eee oe, "Chemotaxis by the Nematode Caenorhabditis elerans: Tdentificat jon of Attrac- tants and Analysis of the Response oy Use of hivcants", S. Ward, PNAS 70, 817- --, , 821 (1973). Known behavior mutants of this nematode 1 were put onto gradients of an: attractant on agar plates covered with egarose beads or sephadex beads. ‘The i patterns resulting differed between the wild type and the mutants Some studies were done to understand the chemotaxis. The hunt for more mutants was. proposed. Clearly, in hunting for mutants, the more worms to be exemined the better. ‘The worms are small enough to be inoculated in 0.05 ml of licuid from an Eppendorf ; pipette. The patterns are formed quickly and photograph well. Analysis of the path can be done by computer in the same way Berg follows the three-dimensional path of E. coli. , . og we ee 26) Further Automation Instrumentation Development. 4 Although the Dumbwaiter and all of its ancillary equipment is expected to be in full operetion when this program-project would begin in June 1975, a number ‘ of specialized accessory instruments wae probably be needed as the biological : provram develongs. A sample of cuch Instrumentation projects that we envision . . ad. ot Pa rs oa . atu hie time Sollovs:. eee nee vee me epee Peete ee Rm eee jp mm square on the agar insteed of the present l0O-rm square. There is a trade-off between the time and cost of photography end the size of agar area covered. Optimizing the trade-off will require different magnif fications for different experiments. he Call Mantpuletion Deviets--Gur orarcent niens ere to use colony mielkors SIG, dint mileneing daevieot su eeclls that have sin ilar phys sical properties. We can well imagine uhge onher elip: vane . ecobites oo aa yee Sat Sos tine, py Fyn} ripen ane aca: vlabkion ayant LO ae Late} nF ee ty Nae tO ead stated ke y date oa ae = Tyce ey . wet ey yy sed onl oa sane re ctres Wabhe Jor colonies of BE. ecti end avaer special call menipwintion doviees wilh be reaubred trom time to ture. Donald A. Glaser Com tinstiow pose 0 LO « Meee UT nt test semis wep emee- -.. Privileged Communication... > . D. Significance . . . m8, , a ee In the discussions.above of the particular ‘biological research projects, the signi- ficance of each one was pointed out. In general, these applications of modern auto- mation technology coupled with corputer-directed pattern recognition and analysis of data offer a poverful new tool for accelerating research in a wide variety of fields of molecular biology and cell biology. They reduce enormously the labor, time, and =: materials required to isolate rate mutants critical at a number of stages in research ; as well as to measure with high accuracy frequencies of genetic and mutational events : which must be known for the genetic dis section of important biological processes. . eee In addition tO the great gains expected in the speed of research in fundanental _, biology, the same large-scale automation techniques offer great promise for a variety | ; Of bio-assay appli¢ations, including the screening of environmental chemicals ror 0 : their potential mutational and caxz rcinogenic cffects; the testing of proposed anti- ; biotics, antineoplastic agents, and cell regulatory substances.” In additional to the : i possibility of large scale testing of chemical agents, it seems possible to make highly accurate ‘measurements of the effects of ionizing, as well as non-ionizing, radiation on a variety of clonable cells. The information resulting is important to studies in fundamcntal biology as well as the difficult problem of setting safe - I “standards for allowable exposure to ionizing radiation among the gerieral population and among workers in industries involving the presence of radioactive substances. : ; With these large scale methods, it may be possible to extend the dose-effect relation- ship gown to very low exposures and- so to discover in an over-all sense whether there | A -, is a threshhold or minimum dose below which repair mechenisis prevent any detectable i ss of} genetic damage at the single-cell level.: - , . Finally, the success of these applications of the cutting-edge of modern technology 7 serves as a demonstration which may stimulate similar applications in industrial ; a :, a8 well as medical and research sectors. We already know of several projects for ‘ ~ ' strain inprovement of antibiotic producing organisms that have been directly stimu- : lated by this work. Representatives of a very large number of pharmaceutical manu- facturing firms, instrumentation manufacturers, and chemical compenies have visited our facilities. Suppliers of agar for.medical and research purposes have also visitcd our facilities and have discussed with us their problems in maintaining uniform repro- ducible quality in their product. Variability is a source of considerable difficulty © in both medical and research applications and we have agreed ina general way to measure batch to batch variations by its effect on colony morphology and growth rates | in an effort to help then anipreve the quality of their product. oo, ftom eee a eerie tere en eee ete The five-year period of this proposed program-project should be ample time to carry through successfully a muiber of the projects we are proposing as well.as to test the feasibility of a number of others and evaluate the usefulness of this kind or ; technology to biomedical science and industry. ee ee oe E. Tacilitics Available oO - : Virus Laboratory = Molecular Biology Department. ee i Many of the biological experiments described here will be developed, at least | i to the pilot stage, in the Molecular Biology Department and Virus Laboratory as has : been done in the past. All of the usual common research facilitics of these labora- ' tories will be available as necessary. In addition, a small, well-equipped machine Shep is av our cisvocal. . se 74 2 eet Me waa s ag an ‘ | | } 3 i ar “e avne eae, me nd oo jot a to | ’ i and vho have oP arene Games Yo neha 2 cme oe i F.- 3 wo Geert es wea i: : . ~" we - . Yr “Conti Eyes foe Privileged Communication - 41. " Tawrence Berkeley Laboratory oO: ‘From time to tine we may call upon special cor ewer te 7 mene “ Donala-1 A. Glaser —— aoe. wwe shops and: consult vith exerts. }-frev the Lavrence Berkeley Laboratory to help us with proziems which they may have already encountered in their High Fner _daboratory. We ‘Blectrical Engineering Departnent scanner are basement o7 and graduate students tae developzent and use of this SYTCGI. eering Department and the Engineering. Research Labore facilities for work of the type we.are undertakings, for those members of Anberested: in applying their special skills located in evecially remodelled *-Campus Computer Center - . Only nodest funds have been, budge ted y Poyzics and -} tion we can ofsen obtain electronic and o ther specialized L-prices and with immediate availability from the excellent s are very fortunate to be able to take advantaze -nical facilities and talent available at the Laurence Ber! keley laboratory. in’Mectrical ingineering have been taki Thus collaboration W rer use .of the Caapus Compu other orosrans. oom facil f the superb tech- AS Tne large-scale automatic equipment including ¢ the computer and flying-spot space provided for that purpose in the the Electrical Rngineering Building, Cory Hall.. Members of the faculty retories offers excellent s @s well as fas special @ unigve opportunity the faculty and greduate students in Electrical Ine gineering and knovledge to biomedical engineering ot . ' - F. ter Center ‘In addi- © supplies at very attractive - ilities cr the , per mene ing an effective role in: ith the Electrical Engin- eee since, until now, we have been able to carry out all the computations associated with our work on Our ovn computing system. magnetic tape and carried ont facilities for large-scale computational WOrk. oo. — Physics Department Laboratory space in the Physics Department When our own system is saturated, we may te eble to reorganize our programs so that some of the pure computation can bé put on at the Campus Computer Center which offers general a ~ is available for this vork it needed and the excellent resources of the Machine Shop and Glassblowing Shop can be used frem time to tine as necessary. Extra-fabrication Space . For fabrication cf much of the sheet me ‘the construction and mcintenance of the large-s granted the use of a corrugated etal building Hall convenient to all of our other crerations. this building was previously used for storing wining shaft. cal cals Loce af Collaboretive Arranzene nts ‘ all of the ecientific inves tigators v visited our racilities Since t relationship ou> oun labor: the precis oak nenbers of that will cevelop betucen atocy remain to be defined. a" PM ae te ce oe eee ae biter eee rn We ene ene Bete tee we we le Le wee sone. Oe Le “. aya ae jaye oe. pe mes noe eee mee , "% and welding work required for 2 automated ecuimsent, we have been ated in the parking lov ‘of Cory Coumonly called the "Ore House", ores obtained from a nearby practice ee ee “We have had extenzive conversation and in some cases correspondence. with vno have proposed projects using our eauipment the Cyclops has been running ' time, we have not encourz sed active york in our laboratory until very tle and igators and these scientii Fic inve in every case sc tentific RAS 8 Fe nee ee at at oe 10, Sete, ee Nee ne mee me eee ee eee ae ee : Bane a as y - : ee see ee only a short Aen er eeren wee: + + eee emmnein Foee e oma ers teh ne tee ee saben ae oe te ee ee net eee ee “investigators proposing projects listed here have independent support for carrying - out these projects in their own leboratories and we intend to provide use of our - "years. Donald A. Glaser oe ot *. : :.. 7 eestor eee gt tebe h - we ee tee oe : ca ao ~- e ~ . ‘ - a i facilities and necessary supplies without any formal arrangements or exchange of funds If scheduling of experiments and assigning of priorities becomes difficult, we will probably invite some of the scientific investigators to join us and constitute an Advisory Committee to help plan the work schedule. It is too early to. foresee accurately how all of these relationships will devélop so no formal administrative. structure for collaboration is being planned at this time. . In addition to the scientific investigators named above, Professor Hervert B. Baskin and Professor Martin Grahem of the Department of Electrical Engineering and Computer Science, University. of California, Berkeley have been very helpful in giving advice concerning computer hardware and software. They gencrously agreed to continue this relationship and perhaps play a more active role in this program in coming G. Principal Investigator Assurance. The undersigned agrees to accept responsibility for the scientific and technical conduct of the research project and for provision of required. | progress reports if a grant is -avarded as the result of this application 9 November 1973 ; mo, . mo TD) cialol Mba Date . Donaid A. Glaser , : . Principal Investigator che \ Vv Privileged Cormunication __.. — #§&» £4 voce eee ee we ees wee ee