SPONTANEOUS AND IONIZING-RADIATION INDUCED MUTATIONS INVOLVE LARGE EVENTS WHEN SELECTING FOR LOSS OF AN AUTOSOMAL LOCUS

The mouse P19H22 embryonal carcinoma cell line contains two distinct c hromosome 8 homologs, one derived from Mus musculus domesticus (M. dom esticus) and the other derived from Mus musculus musculus (M. musculus ). It also contains a deletion for the M. musculus aprt allele, which is located on chromosome 8. In this study, cells with spontaneous or i nduced aprt deficiencies were isolated from P19H22 and examined to det ermine the nature of the mutational events that had occurred. Ultravio let radiation (UV), ethyl methanesulfonate (EMS), and two forms of ion izing radiation, Cs-137 and Cf-252, were used for mutation induction. DNA preparations from the aprt deficient cells were initially screened with a Southern blot analysis and separated into two broad classes: t hose that had lost the M. domesticus aprt allele and those that had re tained it. The overwhelming majority (> 95%) of the spontaneous and io nizing radiation-induced mutants exhibited aprt gene loss, indicating that relatively large events had occurred and that homozygosity for th e deleted region was not a lethal event, Loss of heterozygosity for sy ntenic markers was found to be a common event in cells exhibiting aprt gene loss. In contrast, a majority of the UV-induced mutants (61%) an d a substantial minority of the EMS-induced mutants (38%) retained the aprt gene. A sequence analysis confirmed that base-pair substitutions were responsible for this class of mutation. Gene inactivation associ ated with hypermethylation of the promoter region was found to be a ra re event and was not induced by any of the mutagenic agents tested. Th e results demonstrate the suitability of the P19H22 cell line for muta tional studies, particularly those that are large in nature.