Comparison of the Frequencies of Spontaneous and Chemically-induced 5-bromodeoxyuridine- Resistance Mutations in Wild-type and Revertant

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations vaned with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxyuridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 1G-6 / cell / generation) and low in the wildtype cells (< 3.5 x 10-8 / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed. MOST genetic studies of a specific locus in cultured mammalian cells have involved the X-linked hypoxanthine-guanine phosphoribosyl transferase locus (SZYBALSKI and SMITH 1959; LITTLEFIELD 1963; CHU and MALLING 1968, CHU et al. 1969; ALBERTINI and DE MARS 1970; MORROW 1970; GILLIN et al. 1972). The study of an autosomal locus using the methods developed for 8-azaguanine resistance analysis should provide further information concerning the effect of gene multiplicity on genetic stability (HARRIS 1971 ; MEZGER-FREED 1971 ) . Also, by this method the effect of gene multiplicity on chemically-induced mutation rates can be studied by comparison of heterozygous revertant clones to the homozygous wild-type cells. However, many established cell lines are aneuployd and the presence of the two homologous chromosomes bearing the marker under study can rarely be assumed. Genetics 77 : 309-322 June. 1974.