Induction of multilocus mutations at the Tk1 locus after X irradiation of L5178Y cells at different times in the mitotic cycle.

TK1+/- L5178Y-R16 cells were separated into G1, S and G2/M-phase populations by centrifugal elutriation and were treated with 1.5 Gy X radiation. Cells irradiated in the G1 and G2/M phases were most sensitive to the cytotoxic effects of radiation, while cells irradiated in the G2/M phase showed the highest mutant frequency at the thymidine kinase (Tk1) locus. DNA isolated from independent TK1-/- mutants was analyzed for loss of heterozygosity (LOH) at the Tk1 locus and two microsatellites, D11Mit48 and D11Nds7. Homogenates of each mutant were assayed for activity of galactokinase (GLK), the product of the galactokinase (Glk) gene neighboring the Tk1 gene on chromosome 11. Irradiated G1-phase cells had the highest percentage of mutants showing no LOH. The frequency of mutants with LOH at both Tk1 and D11Nds7 with no loss of GLK activity was high in all cell populations: There was no significant difference in the observed frequency of these mutants between the populations. The frequency of mutants losing GLK activity was low, particularly in cells irradiated in the S or G2/M phases. The possibility that the loss of GLK activity is not indicative of LOH at the Glk gene under the conditions of the present experiments is discussed.