December 1, 1951 Annuel Progress Report and Proposal for Contract Extension to Atomic Energy Commission Contract No. AT (ll-1)-64 Project 10 CYTOGENETIC EFFECTS OF RADIATIONS ON BACTERIA Department of Genetics, University of Wisconsin Project Leader: Joshua Lederberg, Ph.D., Associate Professor of Genetics PROGRESS REPORT The general purpose of this investigation is to analyse the effects of radiations on diploid cells of bacteria (Escherichia coli). This is ootentially favorable material for detecting various types of genetic and cytological changes which may be involved in radiation damage to cells. The present work was entirely preliminary, and designed to establish whether the study should be continned. The scale of the work to date has been extremely modest, expenditures under the contract having been confined essentially to part-pay- ment of the salary of one graduate student resezrch assistant during the pzst five months, Although much of this time was necessarily spent in adapting and learning of certain technicues, the project leader feels that the results point to the desirability of continuing the project on a modest scale. For reasons of technical convenience, the preliminary experiments have been conducted principally with ultra-violet light. Entirely compareble results have been obtained with X-rays, and it mey be vointed out that very marked effects have been detected with doses of 1000 roentgens, at which there is very little reduction in viability. It should be possible to detect genetic effects at dosages of the order of 100 roentgens, Although this is not an unusually small dose for experiments with higher organisms, most microbiological experi- ments have required doses measured in the tens or hundreds of kiloroentgens,. The most prominent effect of ultra-violet treatment of diploid cells is to induce segregation at a high rate. The result is that a treated cell ives rise to a colony which consists mostly of heploids, but usually retains a diploid residue at the center. This is quite the contrary of what would be expected if recessive lethal mutations played a large part in radiation damage. In the diploid cell lethal changes would be masked in the heterozygous condition, but should prevent the develoument of haploid cells carrying the lethal. Thus, recessive lethals should effectively prevent segregation, whereas irradiation seems to induce segregation, even at very low doses. At first, the residual diploid component of colonies from treated cells was overlooked, and it was thought that the unit target of radiation damage could be defined as the haploid-chromosome-set (as has since been suggested by other workers). The picture is greatly complicated, however, by the fact that single treated cells appear to give diverse progeny: induced haploids, and residual diploids. It was then found that the residual diploids were also not all alike: many of them have undergone changes similar to those involved in somaticcrossing-over in Drosophila, This is reflected in the occurrence of new diploid types that are now homozygous for some of the fectors for which they were previously heterozygous. This type of "diploid segregation" has been observed in untreated cultures, but only rarely, whereas a large frection, perhaps the majority, of residual diploids are so affected. The complexity of the genetic changes is paralleled by the cytological findings, Figures 1 and 2 show microcolonies from control and ultre-violet treated cells. A gocd deal of radiation microbiology work so far carried out has assumed that single cells behave as units to radietion response: for example the effects of ultre-violet and X-rays in ceusing delay in growth have been interpreted on the basis of the progeny, taken as a whole, of single cells. Newcombe and Scott have pointed out the difficulties that may arise from failing to take into account the discrepancies in recovery times of different cells. These are mult- iplied by the finding that the early progeny of a single treated cell may then grow at widely different rates, and give different types of genetic change. Much of the complexity in the anelysis of apparent delay in the mutagenic effects of radiations may result from this variable response. The proper analysis of these complex genetic effects reouires the separa- tion of single treated cells under the microscope, followed by a pedigree or microcolony analysis. Only in this way can the distribution of effects in the progeny be separated from selective growth of different tyne of clones. The micromanipulatory technicue necessary for these experiments has just been learned in this laboratory, and has been applied to microcolonies of untreated (control) diploids. Previous susvicions that selective srowth of certain seg rezant types csused « serious perturoation of segregation retios have been con- firmed. The discovery by Lwoff and Gutmann that rediations would activate latent intracellular viruses of bacteria has suggested another modality of rediation effects. As most of our diploid strains are lysogenic, the activation effect might have been suspect as the basis of the genetic and cytological changes, However, preliminary experiments with nonlysogenic lines have given the same picture of responses to ultraviolet light. The genetic effects of ultraviolet light and X-rays are closely simulated by a variety of chemicals, especially hydrogen peroxide end formaldehyde. Of these, however, only the ultra-violet effect is subject to photoereactivation with intense visible light. PROPOSALS FOR FURTHER WORK It is proposed to continue and develop the investigations initiated in this vroject. The single cell isolations with the micromanipulator will be continued to give ea clearer picture of the sequence and kinds of genetic alterations following treatment with radiations. A further search for lethal mutations should be made: no evidence of their occurrence in this material has yet been found. The cytological and genetic effects of X-rays should be com- pared in detail with tnose of ultreviolet light, hydrogen peroxide, formaldehyde, and other mutagens, Finally, the responses of cells with and without latent viruses should be compared to determine under what conditions the activation of latent virus is important in bactericide, and to what extent this activetion can be related to the other cytogenetic effects of the rediations, PERSONNEL Preject Assistant: Ethelyn R, Lively, M.S. 1950. Has conducted cyto- logical work, and more recently, single-cell isolation with micro-manipulator. Project Assistant to be appointed: Melvin L. Morse, M.S., formerly Junior Biologist, Oak Ridge National Leboratory. PROPOSED BUDGET (March 1, 1952 to February 28, 1953) Salary (M. L. Morse, 1/2~time) Project Assistant $ 1320 Supplies, expendable (glassware, reagents) 500 Travel (to use facilities not available at this campus) 200 2020 Overhead, at 8% 162 Total $ 2182 The expected duration of the project, if continued, is to July 1954, Depending on the progress of the work, future reauests may be increased to a level of $4-5000 per annum if a greater concentretion of effort becomes advisable. The principal contrioution of the University of Wisconsin will be the salary of the Preject Leader, end the facilities of a microbiologicel research laboratory. The University is conducting a program of research in the genetics of microorganisms at this laboratory, many phases of which interloc with radiation prceblems, The pudget for the laboratory (including salsries) is approximately $25,000 per year. Approximately half these funds are secured from outside the University, principally from the Rockefeller Foundation, the National Institutes of Health, and the Chemical Corps. These funds are earmerked in very general terms for research in bacterial genetics. RESPONSIBILITINS Or PROJECT LEADER The statement previously filed still applies, except that the laboratory staff has increased to include four to five graduate research assistants, in place of three: two postdoctoral research associates in place of one, and a fulltime technical assistant. Approximately 15 percent of the Project Leader's time will be related directly to this project, although almost all of the bacterial genetic research conducted in this laboratory is more or less indirectly connected with it.