Spontaneous Mutation Rates in Mammalian Cells: Effect of Differential Growth Rates and Phenotypic Lag

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We calculated a spontaneous rate of 26-37 X 10(-6) mutations per cell division for L5178Y MOLY (mouse lymphoma) cells at the thymidine kinase locus (tk(+/-) -> tk(-/-)) using a procedure that isolated and segregated cells during expression. This rate was 50 times higher than when cells expressed the mutant phenotype in suspension. The higher mutation rates obtained with the in situ procedure suggest that many of the mutants, whether expressed or unexpressed, grew more slowly than wild-type cells prior to selection with trifluorothymidine (TFT), implying that the slow growth phenotype is expressed earlier than the TFT(r) (TFT-resistant) phenotype. The loss of mutants was not restricted to cells forming small colonies; the mutation rate for cells forming large colonies was more than ten times higher using the in situ procedure. In this new procedure, the cells expressed spontaneous mutations while growing in semisolid medium for up to 3 days without TFT. Mutants were then selected in situ by adding an overlay of TFT and the visible colonies were analyzed after 11 days. Cells with spontaneous mutations at the tk locus required approximately 30 hr for the more rapidly expressing cells with new mutations to be detected. Most of the TFT(r) colonies selected after 60 hr of growth in semisolid medium represented independent mutations that had accumulated during the first 30 hr. Fluctuation assays were performed both by starting multiple small cultures in suspension followed by growth in semisolid medium using the in situ procedure, and by recalculating the results of the in situ method using the P(0) method; these gave rates of 26 and 37 X 10(-6) mutations per cell division, respectively. Procedures that do not recover slowly growing expressed and/or unexpressed mutants and that ignore the effect of phenotypic lag substantially underestimate the spontaneous mutation rate at the tk locus.

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