HAAS, ET AL.: MUTATION INDUCTION IN BACTERIA 159 



of mutation obtained on M agar might be due to inability of the 

 induced reversions in these tryptophan-deficient strains to be pheno- 

 typically expressed without some initial supplementation. If true, this 

 behavior would indicate that mutation expression requires macro- 

 molecular synthetic events following those involved in gene repli- 

 cation and separable from the former when studying prototrophic 

 mutations in an auxotrophic strain. The results of experiments to 

 test this hypothesis have been previously reported (9). 



A typical CMP challenge experiment was performed; also at the 

 same time, samples were plated on M agar as well as on broth- 

 supplemented M agar (Figure 5). As usual MF closely follows RNA 

 synthesis. However, when identical aliquots are plated at the same 

 incubation times on M agar, few mutations appear until after 40 

 minutes of incubation. At this point MF has practically been com- 

 pleted. After 40 minutes incubation, and closely following DNA 

 synthesis, the mutation frequency obtained on the M agar plates rises 

 sharply, and after 90 minutes the frequency of induced mutation 

 measured is the same on both plating media. The results indicate 

 that a period of protein synthesis subsequent to DNA replication is 

 necessary for expression in the case of auxotrophic reversions. This 

 hypothesis is susceptible to further testing. If the irradiated cells are 

 incubated in complete medium for a time sufficient to allow initiation 

 of DNA synthesis and then CMP is added to the incubating culture, 

 mutation expression should be prevented in that portion of mutant 

 cells which have not yet synthesized the requisite protein. This late 

 CMP treatment should not promote MFD, however, since mutation 

 fixation would already have been completed (see Figure 5). When 

 such an experiment was performed, it was found that if CMP is added 

 after 60 minutes incubation in complete medium, DNA synthesis 

 continues in its presence; but further mutation expression is pre- 

 vented when the cells are plated on M agar. When the culture is 

 treated with CMP after 100 minutes of incubation and then plated 

 on M medium, the mutation frequency is the same as that obtained 

 in an untreated twin culture plated on broth-supplemented M agar. 

 Other experiments were performed with an auxotroph requiring 

 thymine, methionine, and tyrosine (E. coli strain 15 T - Me -Tyr-)- The 

 mutation followed was reversion of the tyrosine requirement. When 

 this strain is deprived of thymine but not amino acids during post- 



