Ultraviolet light-induced mutation and deoxyribonucleic acid synthesis in Escherichia coli.

Exposure of a bacterial population to a mutagenic dose of ultraviolet light generally does not establish phenotypically mutant cells immediately. Rather, a period of subeultivation of the irradiated cells is required before newly induced mutants can be detected (Demeree, 1946). It has been postulated (see Witkin, 1956, for a recent discussion) that the delay between irradiation and phenotypic expression can be accounted for by the time required for three processes: (1) the establishment of a stable, heritable alteration of the genotype, (2) the segregation, if necessary, of the mutant genome from the influence of dominant, nonmutant elements, and (3) the "expression" of the mutant genotype in an altered phenotype. This hypothetical fractionation of the over-all delay is supported in part by the finding, immediately after irradiation, of an interval of instability with respect to the potentiality for mutation. During this period it is possible to influence the ultimate yield of mutants by appropriate manipulations. This interval is, in some cases, demonstrably distinct from the period of growth still required for the completion of phenotypic expression (Lieb, 1959). During this labile phase the mutagenic effects of ultraviolet light can be reduced or photoreversed by exposure to white light. Cultivation of the cells in the dark leads to a fixation of the mutagenic effect with loss of susceptibility to photoreversal (Matney, Shankel, and Wyss, 1958; Doudney and Haas, 1959). Witkin (1953, 1956) and Berrie (1953) have shown that the yield of mutants is also depressed by unusually high or low incubation temperatures. The period of temperature sensitivity is relatively short, lasting about one-third of the postirradiation lag phase. Witkin (1956) has clarified the long-known dependence of the mutational process upon postirradiation nutritional supplementation (Demerec, 1946; Demerec and Cahn, 1953). She has shown that efficient mutation induction requires, or at least is associated with, a high rate of protein synthesis immediately following irradiation. This conclusion rests upon the findings that a full complement of amino acids is the essential nutritional element required and that exposure to chloramphenicol, known to be a specific inhibitor of protein synthesis, depresses the yield of mutants. Again, these factors exert. their influence over only a short time period following irradiation. After this time the mutant yield is fixed and no longer susceptible to change by prolongation or cessation of treatment. Haas and Doudney (1957) and Doudney and Haas (1958, 1959) extended Witkin's findings to a better understanding of the kinetics of the amino acid and chloramphenicol effects. In addition they have discovered that certain effects upon the mutation yield are brought about by preand postirradiation incubation in the presence of various purine and pyrimidine nucleosides and their structural analogues. They interpret their findings as suggesting a role for ribonucleic acid (RNA) synthesis in the process of ultravioletinduced mutation. Subsequently, Witkin (1958) has shown that the duration of the period of amino acid dependence and chloramphenicol sensitivity increases linearly with increasing doses of ultraviolet light. A similar extension of the sensitive period may be produced by incubation of the irradiated cells in caffeine. On the basis of the known ability of ultraviolet light to block deoxyribonucleic acid (DNA) synthesis in Escherichia coli strain B/r, the bacterium used, and the fact that caffeine is an inhibitor of nucleic acid synthesis, Witkin reasons that the termination of the period of mutational lability involves nucleic acid and probably DNA synthesis. Thus, synthesis of RNA, protein, and DNA have all three been implicated in the immediate postirradiation events in a fairly specific manner. These events serve, on the one