-A PROGRAM IN GENETICS AND MOLECULAR BIOLOGY Genetics Department Stanford University School of Medicine October 1968 - October 1973 Submitted by: fo ) f L a { Ne 5 \ was Pea VW Pb ae VO oe Ne PE eet 7 Aoshua Lederberg, Principal Investigator / Department of Genetics _ ‘Stanford University School of Medicine Palo Alto, California This application is intended as a continuation of Grant Number GB 4430 (expiring 10/15/68) entitled "A Program in Genetics and Molecular Biology". A PROGRAM IN GENETICS AND MOLECULAR BIOLOGY The development and expansion of the Genetics Department in its new facilities in the Clinical Sciences Research Building of the Stanford Uni- versity School of Medicine, occupied in March 1966, has been greatly aided by funding from the current "Program in Genetics and Molecular Biology" -- National Science Foundation Grant No. GB 4430. The main emphasis of this grant has been the provision of major items of equipment whose use is com- monly shared by all members of the Department. The greater flexibility which has derived from this type of departmental grant compared to the individual research grants has enabled us to operate much more efficiently and to respond rapidly to equipment needs as research programs change and develcep. The success of the program as an essential complement to individual funding can be judged from the variety of advances documented in the depart-~ mental bibliography. As the program has continued, and many, if not all, of the equipment needs have been met, the Department has been evolving other ccoperative efforts to provide for the maximum and efficient utilization of our facilities. It has become increasingly clear from this that there is a considerable need for personnel and services which operate at a departmental rather than individual level and it is mainly for their provision that the present continuation application is made. Many areas of our individual research programs converge on common techniques and facilities, yet often no one individual is in a position to justify the entire use of a: Professional Assistant or laboratory facilities for these particular needs. The operation of the departmental amino acid analyzer purchased from funds of the present Program is an excellent example. Again the ordering and disposal of supplies and the day by day maintenance of equipment and common wash-up and steriliza- tion facilities is most effectively accomplished by a Departmental Laboratory Coordinator. The faculty group participating in this application consists of: Professor J. Lederberg, Executive - Genetic chemistry of bacteria. Com- puter system for biochemical analysis. Professor W. Bodmer - Genetic chemistry of transformation. Somatic cell genetics and genetics of human white cell antigens. Population genetics. Professor E. Shooter - Molecular neurobiology. Chemical ontogeny and polymorphism of nervous system proteins. Nerve growth factor proteins. Associate Professor L. Herzenberg - Immunogenetics and somatic cell genetics. Assistant Professor A. Ganesan - Mechanisms of genetic recombination. In vitro synthesis of transforming DNA. Senior Research Associate E. Levinthal - Instrumentation research. Senior Research Associate B. Halpern ~ Reagents and techniques for ultra- microanalysis. In addition, a number of research associates of senior stature are con- nected with these and other programs, including Dr. S. Liebes (physics of mass spectrometry). The substantial engineering developmental program in automated instrumentation under N.A.S.A. auspices continues to converge with the main research areas of the department, the development of an automated cell separator being one excellent example of the collaboration. The Exchange Program in Genetics and Molecular Biology between Stanford University and the University of Pavia, funded in part by NSF, is also now in operation and the following Pavia faculty and students are, or will shortly be, working in the Genetics Department: Professor L. Cavalli-Sforza, Drs. M. Polsinelli, S. Barlatti and A. Cefferi and P. Pignatti. Professors E. Glassman of the University of North Carolina and Sarane T. Bowen of San Francisco State College will be spending their sabbatical leaves in the department in 1968-69. A detailed budget for the first year of the program is attached, as well as a general breakdown of expenditures for the subsequent second, third, fourth and fifth years. NATIONAL SCIENCE FOUNDATION Washington, D. C. 20550 RESEARCH GRANT BUDGET SUMMARY Institution: Principal Investigator: Program Name: Stanford University Joshua Lederberg A Program in Genetics Duration: 10/15/68 to Grant Number: School of Medicine Genetics Department and Molecular Biology 10/15/69 NSF Funded Grantee Man Months Man Months (Cal) (Cal) NSF Grant Grantee Share A. SALARIES AND WAGES 1. Senior Personnel a. Principal Investigator Joshua Lederberg 1 3,369 b. Faculty Associates W. Bodmer, A. Ganesan, L. Herzenberg, E. Levinthal, E. Shooter 2. Other Personnel: Technical a. Administrative Officar D. Stuedeman 4 4,000 b, Laboratory Coordinator M. Thomas 12 7,600 c. Professional Asst. to operate Dept. Amino Acid Analyzer V. MacPhee 12 8,400 d. Professional Asst. to operate Dept. Tissue Culture Lab 12 8,400 e. Lab. Technician for Dept. Animal House F. Rodriquez 12 6,600 f. Machinist 2 2,000 Total 37,000 3,369 B. FRINGE BENEFITS (11.6%) 4,292 391 Total 41,292 3,760 C.PERMANENT EQUIPMENT 1. Large liquid Ny tank 5,000 2. Spinner culture apparatus 3,000 Total 10,000 D. EXPENDABLE SUPPLIES AND EQUIPMENT Materials for use in departmental facilities, e.g. wash up room and sterilization, Media Roon, Animal Room; chemical and glassware for depart- mental stores; reference materials and library 7,000 RESEARCH GRANT BUDGET SUMMARY (continued) NSF Grant Grantee Share E. OTHER DIRECT COSTS Maintenance on equipment (purchased on previous grant) 2,000 Minor laboratory rearrangements 2,000 Rent of two complete terminals for use with ACME 4,500 Computer time for batch processing on campus computer facility at $400/hr. 2,000 Total 10,500 F. TOTAL DIRECT COSTS $ 68,792 3,760 G. INDIRECT COSTS (57% S&W) 21,090 1,920 H. TOTAL COSTS 89,882 5,680 REQUESTED FROM NSF 89,882 Stanford University School of Medicine A Program in Genetics and Molecular Biology BUDGET FOR 2ND AND SUBSEQUENT YEARS - 2nd PERSONNEL 43,300 EQUIPMENT 10,000 EXPENDABLE SUPPLIES 7,000 OTHER 10,500 DIRECT COSTS 70,800 The figures for Personnel have been increased by 5% each year. 3rd 45,465 10,000 7,300 11,000 73,765 4th 47,738 10,500 7,600 11,500 77,338 5th 50,125 10,500 7,900 12,000 80,525 Justification for Budget A. Personnel As the research programs of the various faculty members have developed and expanded it has become clear that they overlap in many areas, e.g. in protein sequence work and tissue culture, and we have therefore set up or are setting up such facilities on a departmental rather than individual basis. This provides for the most efficient utilization of facilities and equipment and also, in instances where a person's needs are relatively small (but vital) and do not justify individual requests for funding, for the greatest flexibil- ity in meeting specific research needs. The department has operated an amino acid analyzer on this basis with great success for the past nine months and the budget therefore requests continuing stipend for a Professional Assistant to run this instrument. Three faculty members at the present use tissue culture and the operations again are on a scale that coordination of equipment and media would be of great benefit. It is envisaged that a Professional Assistant would be in charge of such a coordinated effort and that this person would have also sufficient capability to assist each of the faculty groups as required. The use of the Genetics Department Animal Facility has increased far beyond the load for one person and a second Animal Room technician was recently hired. In that much of his work is for general members of the Department rather than one Faculty member, his stipend is now included in the departmental application. Two positions are listed for a part salary for the Senior Laboratory Coordinator and full time salary for his assistant. Between them they take care of the routine management of the departmental facilities and its technical staff, the organization of the wash and sterilization facility, much of the ordering of supplies and equipment and any research that this may involve, and generally the day to day interactions with the service engineers of the building. A continuing part time salary for a machinist is also included in this application. His services in repairing and modifying equipment and installing minor laboratory fittings continue to be invaluable. C. Equipment This category is now much reduced in level compared to the current grant and will be used as indicated to buy, again, equipment for departmental use, for example, for cell storage and culture. D. Expendable Equipment and Supplies These items include materials for use in the common wash up and steriliza- tion facility, the animal house and for departmental stocks of glassware, scintillation vials and chemicals and solvents used in relatively large amounts. Also it includes funds for the purchase of necessary reference materials (e.g. Automatic Subject Citation Alert, ASCA) and other library supplies. E. Other Direct Costs With the availability of ACME (Advanced Computer Facilities for Medical Education) this Department makes large scale and effective use of these major facilities. The cost of the rent of two of our ACME terminals is included in this category as is also computer time for the overnight batch processing of some of our larger requirements. Existing Facilities The department covers some 11,000 square feet in the new Clinical Sciences Building of the Stanford Medical Center complex providing integrated areas for microbial, cell and human genetics, molecular neurobiology and computer work. Common facilities for wash up and sterilization are available as are hot and cold room facilities. The Instrumentation Research Laboratories are housed in 6,000 sq. feet of appropriately designed areas on the Ground Floor beneath the Genetics Department. The department is equipped with all the usual apparatus for work in the fields outlined above. Major items of equipment include: Tricarb scintillation spectrometer * Spinco analytical ultracentrifuge Spinco and IEC preparative centrifuges Spinco amino acid analyzer Zeiss spectrophotometer Bendix and Quadripole mass spectrometer with interfaces to the IBM 360/50 computer Existing Funding. 10. No other funding matches the purpose of this application, however, component elements of our investigations are supported as follows: From National Institutes of Health Grant No. AI-5160 GM-14108 GM-14650 NB-04270 GM-12075 CA~04681 GM-295-10 Application Pending GM-35002 Grant Title Genetics of Bacteria DNA Synthesis & Genetic Recombination Genetics of Human Tissue Antigens Molecular Neurobiology Genetics of Immunoglobulins Genetic Studies with Mammalian Cells Final year of current period of Training Program Renewal of Training Program in Genetics Career Award: Dr. Walter F. Bodmer From National Science Foundation GB 5862 GB 6878 Genetic Chemistry of DNA Mediated Bacterial Transformation Subunit Structure of Nerve Growth Factor Dates 9/1/64- 8/31/68 6/1/66- 5/31/69 12/1/66- 11/30/69 12/1/67- 11/30/70 6/1/64- 5/31/69 9/1/67- 8/31/72 7/1/68- 6/30/69 7/1/69- 6/30/74 1/1/67- 12/31/71 12/1/66- 11/30/68 9/1/67- 8/31/69 Direct Costg _ $ 253,000 104,300 124,000 202,000 149,500 272,000 115,000 756,650 120,000 76,950 44,500 li. From National Aeronautics and Space Administration NASA NsG 81-60 Cytochemical Studies of Planetary 9/1/67- 410,000 Microorganisms 8/31/68 (Instrumentation Research Lab.) 12. Joshua Lederberg - Genetics of Bacteria The DNA transfer system ("transformation") in Bacillus subtilis offers a particularly favorable opportunity to study the physical and chemical properties of DNA molecules in relation to their biological activity. Over the past several years my principal co-investigators have been Professor Walter F. Bodmer and Professor A. T. Ganesan. Their work now has become well differentiated and receives independent support, but the studies summarized in the attached publi- cations were initiated during the current term of the grant. The main questions we have examined have concerned: The linkage system, as manifested in DNA transfer; The fractionation of biologically active DNA, assigning different genetic activities to different average base compositions; The effects of fragmentation of DNA molecules by physical shearing, and by nuclease attack; The mechanism of integration of DNA in the course of the transformation process; The association of the DNA replication process with a cell-membrane bound polymerase. In collaboration with Professor Arthur Kornberg and other members of the Department of Biochemistry we had also attempted to demonstrate the replication of biologically active DNA by E. coli DNA polymerase. Those experiments were, however, unsuccessful ~- in considerable part, as we now know, because of our ignorance of polynucleotide ligase. The main impact of these studies has been to verify the correspondence of the properties of genetic activity of DNA with distinctive sequences of nucleo- tides, and to provide some further technical facilities for the ultimate descrip- tion of genetic information in chemical terms. During the period covered by this report many workers have entered similar fields and the literature now contains many examples of the building of new edifices on previously established foundations. 13. I propose now to concentrate on the natural or experimentally induced occurrence of insertions and inversions in B. subtilis. In enteric bacteria, mapping studies have shown a remarkable congruence of gene sequences among bacterial species which have already diverged significantly in DNA homology as tested by hybridization-reanealing experiments. Conservation of gene order is also evident in the organization and persistence of operons, showing coordinate repression; these are frequent in bacteria but rare in higher forms. Most or all of the examples of inversions found experimentally in bacteria can be explained either as methodological artifacts (1) or as consequences by crossing over of episome-bound segments with redundantly homologous segments of the whole DNA (2). These observations were vague forerunners of the now accepted understanding that bacteria differ from eukaryons in 1) The simple organization of the chromosome as a single polynucleotide sequence, and 2) The absence of any common mechanism for "holo-ligation repair" of the chromosome. The recent clarification of the dark repair of single-strand breaks in DNA (reviewed in 3) has pointed up mechanisms of template-directed repair (hemiligation) in bacteria. These are fundamental to recovery from UV-damage, to recombination mechanisms, and probably to the post-editing of newly replicated DNA. There is, on the other hand, no good evidence for any normal mechanisms for joining or rejoining broken DNA strands except for hemi-ligation, i.e., when the broken strand is. entwined with a complementary template strand that spans the broken ends. It may be previous to define a process that may not exist, but we are then looking for holo-ligation, i.e., some way to join broken strands, or double helices, without the help of a homologous template. The potential extent and limitations of holo-ligation relate to many current problems in chemical genetics and cell biology. These include, for example: 14. 1) Genetic variation in recovery from X-ray damage and the mechanism of recovery from double-strand scissions. 2) A sharper confrontation with chromosome structure of eukaryons in which broken chromosomes can indeed be rejoined (but is this a covalent nucleotide assembly?) 3) Transcription problems: framing, the reading of inverted sequences, and strand selection. 4) The barrier to promiscuous recombination of DNA from different sources. Point 4 may be the most far-reaching for every level of genetic manipulation, both investigative and applied. The rejection of foreign DNA by competent Bacillus subtilis cells may illustrate one evolved mechanism whereby a cell has been able to protect itself against unlimited intrusion by foreign genes, arising by chemical scrambling or by virus infection. The research utility of freely moving genes from another species into bacteria needs no elaboration, e.g. for the study of transcription-control, and to facilitate analyzing the genetic competence of DNA from differentiated tissues of higher organisms. Important practical utilities would follow from the incorporation of human genes into suitable cryptic virus DNA for kind of transductional therapy for human genetic disease (4). References: 1. Glansdorff, N., 1967. Pseudoinversions in the chromosome of Escherichia coli K-12. Genetics 55:49-61. 2. Berg, Claire M. and Roy Curtiss III, 1967. Transposition derivatives of an Hfr strain of Escherichia coli K-12. Genetics 56:503-525. 3. Hanawalt, Philip C., 1968. Cellular recovety from photochemical damage. Ch. 11, Photophysiology, Vol. III (A. C. Giese, ed.) Academic Press, New York. 4. Rogers, Stanfield, 1966. Shope papilloma virus: A passenger in man and its significance to the potential control of the host genome. Nature 212:1220-1222. Personnel Postdoctoral: S. Barlati M. Polsinelli Predoctoral: H. Eisenstark I. Majerfeld W. Spiegelman L. Okun 15. Polysome aggregation in B. subtilis Genetic translocation in B. subtilis Conditional lethals in B. subtilis; texicity of DMSO Adding homopolymer terminations to B. subtilis DNA Regulation of lambda bacteriophage High molecular weight transforming DNA Education: 1938-41 1941-44 194k 1946-47 Experience: 1945-6 1946-47 1947-59 1950 1957 1957-59 1959- 1961- Special field: Distinctions: 1957 1958 16. JOSHUA LEDERBERG Department of Genetics Stanford University School of Medicine Palo Alto, California Phone: 94304 (415) 321-1200 Ext. 5801 Stuyvesant High School (New York City) B.A., Columbia College Enrolled as medical student, Columbia University College of Physicians and Surgeons Ph.D., Yale University. Se.D. (h.c.) 1960; also Wisconsin (1967); Columbia (1967). Research assistant in zoology (with Professor F. J. Ryan), Columbia University Research fellow of the Jane Coffin Childs Fund for Medical Research at Yale University (with Professor E. L. Tatum) Professor of Genetics, University of Wisconsin Visiting Professor of Bacteriology, University of California, Berkeley Fulbright Visiting Professor of Bacteriology, Melbourne University, Australia Chairman, Department of Medical Genetics, University of Wisconsin Professor, Genetics and Biology, and Executive Head, Department of Genetics, Stanford University Director, Kennedy Laboratories for Molecular Medicine, Stanford University Genetics, chemistry and evolution of unicellular organisms and of man. National Academy of Sciences Nobel Prize in medicine (for studies on organization of the genetic material in bacteria) Public responsibilities: 1961-62 1950-.. 1958-.. 1960-.. 1967- Personal Data: b. May 23, President (Kennedy)'s Panel on Mental Retardation President's Science Advisory Committee panels. National Institutes of Health, National Science Foundation study sections (genetics) National Academy of Sciences: committees on space biology NASA committees; Lunar and Planetary Missions Board NIMH: National Mental Health Advisory Council 1925; Montclair, New Jersey 17. Walter F. Bodmer - Genetic Chemistry of DNA Mediated Bacterial Transformation; Somatic Cell Genetics and the Genetics of Human White Cell Antigens; Population Genetics. 1. Genetic Chemistry of DNA Mediated Bacterial Transformation The main aim of this research is to further the understanding of the pro- cesses of integration and recombination during DNA mediated transformation in Bacillus subtilis. Earlier work from our laboratory was instrumental in show- ing that donor DNA was incorporated into small single-stranded regions of the recipient genome (Bodmer and Ganesan 1964) and in establishing the extent to which DNA synthesis may be involved during transformation (Bodmer 1965). This work clearly showed that there is no major amount of DNA synthesis dur- ing uptake and integration of donor DNA, though the amount of repair synthesis that may be involved and the specific enzymatic steps leading to final integra- tion remain incompletely understood. Understanding these problems remains a major part of our research effort. Specific research projects currently under- way or being planned are a) The properties of a mutant which is a genetic rearrangement will be investigated during transformation, with special reference to testing for heterozygosity in the products of transformation. b) Attempts are being made to assess the true extent of repair synthesis during transformation. High specific activity p?? labelling during transformation and 5-Bromouracil labelling during transformation accompanied by physical separation of competent cells, are the two techniques currently envisaged for these studies. c) A double marker transformation assay has been shown to be a likely indicator of "cross-correction repair" during transformation. The 18. effect of UV sensitive mutants on this system are being investigated. d) work with multiply marked recipient strains is continuing with a view to determining further the relationship between DNA integration during transformation and regions of initiation of DNA synthesis. Experi- ments are also planned to test whether protein synthesis is required for reinitiation of DNA synthesis following transformation. e) Further experiments are planned on the early events following uptake of donor DNA during transformation, in particular to determine the nature of the early native material found following addition of donor DNA. In addition, efforts will be made to characterize the transition from non-covalent to covalent association between donor and recipient DNAs. Human Somatic Cell Genetics and the Genetics of Human White Blood Cell Antigens. a) Our laboratory,in collaboration with Dr. Rose Payne of the Department of Medicine, has been instrumental in identifying some of the major anti- gens of the HL~A human white blood cell antigen polymorphism and in interpreting the relationships between the genetic determinants for these antigens (Payne et al 1964, Bodmer and Payne 1965, and Bodmer et al. 1966). During the last 18 months we have developed a convenient microcytotoxicity assay combining Terasaki's micro droplet assay with the technique of fluorochromasia (Bodmer et al 1968). We have used this assay extensively for population genetic and other studies directed towards further development of knowledge of the genetics of the human white cell antigen systems. Current areas of investigation are b) 19. i. We are attempting to devise more sensitive assays for the antigens using anti-human gamma-globulin serum both for cytotoxic and agglutination assays. ii. New antigenic specificities are being characterized by absorption analysis of a number of sera, combined with population and family studies. iii. We have undertaken a major population genetic study, in collabora- tion with Professor L. L. Cavalli-Sforza, of the pygmies of Central Africa and of other African populations. We plan to extend these population studies to other racial groups through collaboration with other investigators. iv. We are investigating the potential uses of cattle isoantisera for typing human white cell antigens and the general question of species cross-reactivity with respect to these antigenic systems. During the last year we have initiated a program of research in human somatic cell genetics. This is based on using the technique of cell fusion mediated by inactivated Sendai virus. Hybrid lines are being made by fusing human peripheral blood lymphocytes with base analogue resistant mouse cell lines derived from known inbred mouse strains. Hybrids are initially recognized by their chromosome constitution. A major source of genetic markers are the white cell antigens being studied in our laboratory. A micro-mixed agglutination technique for the identification of these antigens on cell cultures is being develop- ed and is being applied to study the distribution of these antigens on our human-mouse hybrids. Other genetic markers will, of course, be 20. studied, in particular electrophoretically distinguishable variant enzymes. The karyotypic and genetic constitution of hybrid lines is being studied and attempts are being made to correlate the presence of combinations of human genetic markers with the chromosomal consti- tutions of the hybrids. These studies will be coupled with attempts to obtain control changes in the hybrid karyotype. Other lines of human cells will be used for studies related to the problem of gene expression as a function of differentiated cell type. 3. Population Genetics The emphasis of work in this area is on the theoretical analysis of popu- lation genetic models and their interpretation, particularly with relation to the problems of human genetics. For some years I have been involved in an active program of research in theoretical population genetics with Professor Karlin of the Mathematics Department. This unique relationship has provided an opportunity for combining a sophisticated mathematical approach with direct contact with experimental genetics. Recently, work has been done to extend understanding of models for the Rhesus blood group incompatibility system. In collaboration with Professor L. L. Cavalli-Sforza, new models have been developed to explain observed differences in gene frequency in terms of observed migration patterns, using a migration matrix approach. Further work is planned on the problem of the interaction of linkage and selection, particu- larly with respect to the conditions under which there is selection pressure for tighter linkage and the problems of dealing with more than two loci. In addition, some work has been done on using data on amino acid substitutions to match observed evolutionary rates with those expected from population 21. Walter F. Bodmer - Genetic Chemistry of DNA Mediated Bacterial Transformation; Somatic Cell Genetics and the Genetics of Human White Cell Antigens; Population Genetics. genetic theory. A major survey of theoretical population genetics, under- taken in collaboration with Professor Karlin and Mark Feldman, a graduate student, is nearing completion. Personnel Postdoctoral A. J. Darlington V. Miggiano T. LIha F. Seudo Predoctoral M. Nabholz Research Assistants L. Wang J. Bodmer G. Gerbrandt M. Tripp Genetic chemistry of DNA mediated bacterial transformation Human somatic cell genetics Human white cell antigen genetics Theoretical population genetics Human somatic cell genetics Genetic chemistry of DNA mediated bacterial transformation Genetics of human white cell antigens Ww a vw " WwW Personal Data: Education: 1953-56 1956-59 Experience: 1958-60 1960-61 1961 1961-62 1962-66 1966-68 1968- Special Field: 22. WALTER F. BODMER CURRICULUM VITAE Born: Frankfurt am Main, Germany, January 10, 1936 British citizen, U.S. Immigrant Wife: Julia Gwynaeth Bodmer 3 children Soc.Sec. No. FY B.A., Clare College, Cambridge University Ph.D., Clare College, Cambridge University Research Fellow, Clare College, Cambridge Demonstrator, Department of Genetics, Cambridge University Official Fellow, Clare College, Cambridge Fellow, Visiting Assistant Professor, Department of Genetics Stanford University School of Medicine. Assistant Professor, Department of Genetics, Stanford University School of Medicine. Associate Professor, Department of Genetics, Stanford University School of Medicine. Professor, Department of Genetics, Stanford University School of Medicine. Chemical Genetics, Human and Population Genetics. Public Responsibilities: 1964-67 1964-67 1968- National Science Foundation, Genetics Panel National Institute of Allergy and Infectious Diseases, Committee for Collaborative Research in Transplantation and Immuno logy Associate Editor, American Journal Human Genetics 23. A. T. Ganesan ~ In vitro Synthesis of Transforming DNA, Mechanism of Genetic Recombination The Bacillus subtilis genome replicates sequentially from a fixed origin, as judged by isotopic transfer and gene frequency distributions in transformation experiments. An in vitro system using E. coli polymerase for the replication of DNA from B. subtilis yielded products which were biolo- gically inactive. Thermal denaturation of these molecules was spontaneously reversible. These abnormalities may reflect interruptions and lack of control in coherent replication in the in vitro system. Earlier experiments in our laboratory suggested that in vivo the nascent DNA might be bound to a particulate fraction which also contained DNA poly- merase of high specific activity. Using the nascent DNA already present, the polymerase in the particulate fraction was able to synthesize DNA (Judged by the incorporation of the labeled deoxytriphosphates into cold acid precipi- table material) when supplied with all four deoxytriphosphates and Me". Our approach to the problem of synthesizing biologically active DNA in vitro has been to study the pattern of replication by this particulate fraction, starting from relatively crude preparations and following it through different steps of purification of the polymerase. Careful isolation of the active protein complex free of cell membrane and other components has resulted in a purification of 100 to 150 fold compared to the initial lysate. This preparation has been found to differ in some respects from the highly purified preparations of DNA polymerase from E. coli and B. subtilis. Unlike the latter, our preparation preferred native bihelical DNA to denatured DNA as a primer. Double stranded DNA was 6 to 8 times more efficient as primer than denatured DNA. In our system dAT-copolymer 24. A. T. Ganesan - In vitro Synthesis of Transforming DNA, Mechanism of Genetic Recombination. (continued) was only 2 to 4 times more active as a primer than the native DNA, while with the purified polymerases dAT-copolymer has been reported to be 20 times more active. The enzyme complex sediments with a unimodal distribution in sucrose gradients. Of the ions tested Me** was the most effective in the reaction. Traces of Na’ and K* stimulated the reaction in the presence of Me**. In a reaction, 20% of the amount of primer added was synthesized in 30 minutes. There was no significant amount of exonuclease activity found in the poly- merase. The endonuclease activity associated with the preparation can be partially inhibited by RNA. 15 When transforming DNA labelled with N’’, deuterium and HS was isolated from genetically marked B. subtilis and used as a primer in a reaction of cl4 labelled deoxytri- our partially purified preparation with light (x! H) phosphates, it was possible to demonstrate synthesis of DNA molecules of lighter density as observed by CsCl density gradient centrifugation. 80% of the molecules are denaturable and 90% are rendered into acid soluble mono- nucleotides by E. coli Exonuclease-l1. The products composed of hybrid molecules containing one strand of heavy DNA and one strand of light DNA. The hybrid molecules are biologically active. In addition to the hybrid, 4% of the biological activity was associated with completely light and hybrid. Of these light molecules, at least 10% carry 3 known genetically linked genes, while the majority are only active for single gene transformation. The enzyme complex is presently studied by different physical techniques, to detect various activities, that form the active complex. 25. A. T. Ganesan - In vitro Synthesis of Transforming DNA, Mechanism of Genetic Recombination Personnel Postdoctoral F, Gillin Genetic control of DNA Synthesis in B. Subtilis Predoctoral P. Laipis In vitro synthesis of transforming DNA Research Assistant N. Buckman Mechanism of genetic recombination In vitro synthesis of transforming DNA