MOLECULAR ANALYSIS OF MUTATIONS AT THE TK LOCUS OF L5178Y MOUSE-LYMPHOMA CELLS INDUCED BY ETHYL METHANESULFONATE AND MITOMYCIN-C

Mutations at the tk locus of mouse-lymphoma L5178Y cells were induced by treatment with ethyl methanesulphonate (EMS), primarily a point mut agen and mitomycin C (MMC), a potent clastogen. Mutant colony size was distinctly bimodal with 35% of spontaneous mutants growing as small c olonies and 65% large. The proportion of small colonies increased only slightly to 41% in EMS-treated cultures but to 64% after MMC treatmen t.Mutations were analysed by Southern and Northern blotting. Digestion of DNA with the restriction enzyme, Nco I, revealed that many mutants had lost a 6.3-kb fragment which constituted the loss of the entire t k gene. Almost all of the EMS-induced large-colony mutants analysed (9 /10) retained the tk+ allele suggesting the presence of an intragenic mutation. Of the small-colony mutants, half (6/12) had lost the tk+ ge ne and presumably other genes affecting growth and half retained the t k+ allele suggesting point mutations in both the tk gene and other sit es in the genome affecting growth. A very different spectrum of mutati on was induced with MMC. Only 1/12 of the large-colony mutants were du e to intragenic mutation, the remaining large-colony mutants having lo st the tk+ allele while all the small-colony mutants had lost the tkgene presumably with the deletion extending to genes essential for nor mal growth. Northern blot analysis showed no changes in the size of tk transcript in any mutants. Alterations in the amount of tk mRNA were not detectable since all mutants produced an mRNA of similar size and amount, which may indicate the production of an abnormal mRNA from the tk- allele. Unlike cell-mutation assays that use hemizygous loci (suc h as hprt+/0) for detecting potential chemical carcinogens, the mouse- lymphoma tk+/- assay allows the recovery of both intragenic and interg enic mutations thus enabling the detection of both point mutagens such as EMS and potent clastogens like MMC.