264 



EVELYN M. W1TKIN 



determine the effects of chemicals on the frequency 

 of reverse mutations from biochemically deficient 

 mutants of E. coli are being planned with this need 

 in mind. 



The use of phage-resistance in E. coli as material 

 for the investigation of chemically induced muta- 

 tions offers certain unique possibilities, in addition 

 to its value as a screening test. The analysis of 

 dosage-effect relations, as well as the quantitative 

 investigation of the delayed expression of induced 

 mutations characteristic of radiations and mustard 

 gas, can be carried out with this material. It may 

 also be possible to approach another interesting 

 aspect of induced mutations, the comparison of the 

 effectiveness of mutagenic chemicals on resting and 

 dividing cells. It must be remembered, however, that 

 bacteria are relative newcomers to the laboratory of 

 the geneticist, and that a longer and more intimate 

 acquaintance may be required to establish the re- 

 liability of these organisms as tools for the study of 

 the broader problems of heredity and mutation. 



Summary 



Four compounds were tested for mutagenic 

 activity in E. coli. The techniques used involved 

 suspending resting bacteria in solutions of the 

 chemicals, under conditions resulting in the death 

 of about 99% of the treated cells. The number of 

 mutants resistant to a bacteriophage, Tl, per 10 8 

 survivors of a treated culture was compared with 

 the number per 10 8 untreated bacteria from the 

 same culture. Only mutations expressed phenotypi- 

 cally before division of the exposed individuals 

 were detected. 



The number of mutants per 10 8 survivors was 

 found to be higher in cultures treated with sodium 

 desoxycholate, pyronin and acriflavine than in un- 

 treated samples of the same cultures. No such in- 

 crease was obtained with methyl green. Since mu- 

 tants and nonmutants were shown to be equally 

 sensitive to the toxic action of each of the com- 

 pounds, it has been concluded that selection is not 

 responsible for these results. Sodium desoxycholate, 

 pyronin and acriflavine are considered, therefore, to 

 ue mutagenic, while methyl green is not. 



The number of mutations induced by sodium 

 desoxycholate is directly proportional to the time 

 of exposure to a 5% solution of this compound. 



Acknowledgments 

 The author wishes to acknowledge the helpful 

 suggestions of Dr. M. Demerec, and the efficient 

 assistance of Miss Marion Crippen in many of these 

 experiments. 



References 



Alloway, J. L., 1933, Further observations on the use of 

 pneumococcus extracts in effecting transformation of 

 types in vitro. J. exp. Med. 57: 265-278. 



Auerbach, C, and Robson, J. M., 1944, Production of mu- 

 tations by allyl isothiocyanate. Nature, Lond. 154: 81-82. 



Avery, O. T., MacLeod, C. M., and McCarty, M., 1944, 

 Studies on the chemical nature of the substance inducing 

 transformation of pneumococcal types. Induction of 

 transformation by a desoxyribonucleic acid fraction iso- 

 lated from pneumococcus Type III. J. exp. Med. 79: 

 137-158. 



Brachet, J., 1940, La detection histochimique des acides 

 pentosesnucleiques. C. R. Soc. Biol., Paris 133: 88-90. 



Demerec, M., 1946, Induced mutations and possible mechan- 

 isms of the transmission of heredity in Escherichia 

 coli. Proc. Nat. Acad. Sci., Wash. 32: 36-46. 

 1943, Mutations in Drosophila induced by a carcinogen. 

 Nature, Lond. 159: 604. 



Demerec, M., and Latarjet, R., 1946, Mutations in bacteria 

 induced by radiation. Cold Spring Harbor Symp. Quant. 

 Biol. 11: 38-50. 



Demerec, M., Latarjet, R., Luria, S. E., Oakberc, E. F., 

 and Witkin, E. M., 1946, The gene. Yearb. Cam. Inst. 

 45: 143-157. 



Fieser, L. F., and Newman, M. S., 1935, The choleic acids 

 of certain carcinogenic hydrocarbons. J. Amer. Chem. 

 Soc. 57: 1602-1604. 



Horowitz, N. H., Houlahan, M. B., Huncate, M. V., and 

 Wricht, B., 1946, Mustard gas mutations in Neurospora. 

 Science 104: 233-234. 



Luria, S. E., and Delbruck, M., 1943, Mutations of bacteria 

 from virus sensitivity to virus resistance. Genetics 28: 

 491-511. 



McIiavain, H., 1941, A nutritional investigation of the anti- 

 bacterial action of acriflavin. Bio-chem. J. 35: 1311- 

 1319. 



Mtrsky, A. E., and Pollister, A. W., 1946, Chromosin, a 

 desoxyribose nucleoprotein complex of the cell nucleus. 

 J. gen. Physiol. JO: 117-148. 



Muller, H. J., 1947, The gene. (Pilgrim Trust Lecture). 

 Proc. Roy. Soc. Lon. (B) 134: 1-37. 



Tatum, E. L., 1946, Induced biochemical mutations in bac- 

 teria. Cold Spring Harbor Symp. Quant. Biol. 11: 278- 

 284. 



Wteland, H., and Sorce, H., 1916, Untersuchungen iiber die 

 Gallensauren. II Mitteilung. Zur Kenntnis der Cholein- 

 saure. Hoppe-Seyl. Z. 97: 1-27. 



Witkin, E. M., 1947, Genetics of resistance to radiation in 

 Escherichia coli. Genetics 32: 221-248. 



Discussion 

 Hotchkiss: In examining the most interesting 

 results of Dr. Witkin, I have been struck by the 

 fact that the actual recovery of mutants from a fixed 

 number of original cells, which is the count actually 

 observed in the laboratory, is maximal in the high 

 survival range and decreases markedly as the per- 

 centage of total survivors drops. Since the number 

 of mutants does not however decrease to the extent 

 that the total population does, it tends to rise 

 rather considerably, if calculated over to the basis of 

 survivors. It has been recognized by the author that 

 to view this latter calculated value as a "mutation 

 rate" involves the assumption that the phage- 

 resistant mutants do not have increased survival 

 value in solutions of the chemical mutagens used. 

 Indeed for this reason, trouble was taken to demon- 

 strate that stock phage-resistant mutants do not 

 survive longer than non-mutants in desoxycholate. 



!27 



