UNIVERSITY OF CALIFORNIA PARTMENT OF BACTERIOLOGY ~sRKELEY 4, CALIFORNIA April 10, 1950 Dr. Joshua Lederberg Dept. of Genetics College of Agriculture University of Wisconsin Madison 6, Wisconsin Dear Dr. Lederberg, I would like to thank you for your suggestions regarding changes in my application for an American Cancer Society Fellowship. I made every attempt to incorporate your proposals into the application. I was very much heartened to find that you approve in essence of the program that I have outlined. Out here it is generally believed that an investigation of the role of normal metabolites in spontaneous mutations would lead one on a fool's errand, for "are not Spontaneous mutations caused by slips in chromosome duplication", or "doesn't any chemical that causes death cause mutation", etc. I am looking forward to your visit this summer and to spending next year working in your laboratory. If the fellowships fall through, would there be a possibility of obtaining a job in your laboratory? So far I have been refused the N.R.C. and the Merck Fellowships. Thgewe still remain the American Cancer Society and the U.S.P.H. Service Fellowships. I have just had an interview with Tatum with regard to the American Cancer Society Fellowship. All I can do now is hepe. I was very happy to learn of the results you obtained with CH,0. You were correct concerning the negative results I obtained in inducing mutations of coli B to phage resistance. I had not allowed for phenotypic lag. J am in the process of setting up an experiment where growth of the formaldehyde treated cells could take place before phage infection. Have you performad such an experiment? If so, I would be very much interested in hearing your results. A. Doermann mentioned to me last summer at Cal. Tech. that Witkin reported negative results with CH.0. Are you familiar with this work? Did she also limit herself to so-called “zero-point mutations"? You asked for some details concerning my experiments with CH I would have communicated them to you sooner, but I had planned additional experiments, and I thought I would wait until these were completed. The work was begun in the summer of 1948, just after the work of Rapoport became available, and was left too soon, due to the complications that developed, and also to my desire to broaden out along biochemical lines so as to benefit from the storehouse of bio- chemical techniques and thought available at Cal. 1. A washed suspension of Fseudononas Pluorescens, suspended in phosphate buffer, pH 7, is treated with CHsO (final concentration 0.1%) for 15 minutes. Phosphate buffer is then added, celis are centrifuged down, supernatant liquid poured off, and cells are ~De UNIVERSITY OF CALIFORNIA PARTMENT OF BACTERIOLOGY BERKELEY 4, CALIFORNIA resuspended to the original volume. Aliquots are plated on itaconate mineral agar plates to determine the number of mutants, and the viable count is determined by serial dilution and plating on yeast extract agar. A control tube containing a similar suspension of cells is run together with the experimental tube through all the processes, and instead of the addition of CH,0, a similar volume of distilled water is added. The resuits of a typical experiment follow: Viable count Viable count Per cent No. mutants m/1x10° cells f0.1ce /tube survivors /O.1¢e¢ Sec suspen- sion 28 8 10 1? Control 2.5 x 10 1.26 x 10 100% ie 10 2 he Av. 25 225 After 15 236 m twith 3.4x 10 1.7 x 10 0.014% 255 725 x 10 0.u0¢ CHO 247 AV. 247 2. Itaconate mutants isolated after CHO treatment are not any more resistant to CH,O than the untreated #ild type. 5. Commercial CH,0 contains many impubities. A pure sample of CH,0 was prepared and trisd with similar results. 4. In order to eliminate the possibility that the increased number of mutants may have been the result of additional growth of the parent culture on the itaconate plates, due to traces of CH.0 which were carried over with the cells, the following experiments were conducted: a. Double washings --- sigilar results. b. The mutants were counted by a tube method. This enables one to follow the growth of the parent culture as well as providing additional means of determining the number of mutants present. After CH,O treatment, the washed suspension is diluted out in itaconate mineral liquid medium and tubed so as to place about one mutant in each tube. Growth of the wild type was followed daily in these tubes by platings on Y.E. and itaconate, and turbid tubes were recorded, Turbidity was demonstrated to be the result of itaconate mutation. A similar procedure was followed with an untreated suspension. This experiment conclusively showed that the CH,0 treated cells (wild type) grew to the same slight extent as the untgwéated cells and the mutant count by this method checked with the mutant counts as determined by plating. -Ze UNIVERSITY OF CALIFORNIA _PARTMENT OF BACTERIOLOGY BERKELEY 4, CALIFORNIA 5. An attempt to study the kinetics of the formaldehyde effect revealed the following difficulties: a. Use of different batches of yeast extract, as well as other media, resulted in large variation in viable count after CH,0 treatment, although there were no diffgsrences in viable counts demonstrated with untreated cells: yeast autolysate agar 3 x 10°% survivors - 5% Y.E. agar 0.3% 3% Y.E. agar 22S tt .5% Mineral Asparagine 6.1% rt b. When a mutant count gt CH.0 treated cplis is performed if there are 200 colonies from a 10° dilition, a 10-! Ailution of the Same suspension will revéal only 5, or 2, or sometimes 10 colonies. This Phenomenon may be due to the Gooreage in number of living celis on the 107+ plate, which when present provide the environment for reactivation of itaconate+, induced, deactivated cells, c. If one can rely on the 10° plate to count the mutants, the absolute number of mutants produced reaches a maximum and then falls off slightly with increased concentrations of CHO The relative number of mutantw increases continually. d. A plot of the log of the per cent survivérs against time (0.1% CHO at 5, 10, 15, 20 min.) in about 10 experiments reveals a tremendous variation in rates of killing. All experiments had the following results in common. During the first 5 minutes about 99% of the cells were k&lled and thereafter the remainder were killed at a much slower but constant rate. These results are similar to those of Demerec and Latajet obtained with ultraviolet treatment of resting cultures of coli B. If cells are treated with varying concentrations of CH,O0 (from 0.01% to 0.1%) for 15 minutes, the plot of the log of the per cent survivors against concentration is a straight line. This account, of course, is, of necessity, brief. If it is at all possible, I would appreciate any and all comments you would care to make concerning these experiments. Hoping to hear from you soon, I remain, Sincerely yours, ‘