Deletion mutations and chromosome rearrangements have been implicated in a variety of human genetic defects including oncogene activation. Little is known regarding the mechanisms by which genomic rearrangements arise, largely due to the lack of convenient systems for the study of such events. The Chinese hamster overy (CHO) cell line, AS52, has been demonstrated to be particularly useful in the study of deletion mutations. AS52 cells were constructed by transfecting a single copy of the bacterial gpt gene into a hprt-deficient CHO host and isolating a cell line that carries stably integrated into the CHO genome. AS52 calls appear to be capable of detecting mutations induced by clastogens or radiomimetic agents while analogous loci such as hprt show little or no mutational response to many of these agents. However, classical point mutagene such as UV, EMS, ICR-191 induce mutations at either loci with approximately equal efficiency. These results are thought to reflect the nature of the gpt integration site. We propose that AS52 cells allow the recovery of viable multilocus deletions i.e., deletions affecting all or part of the locus and adjacent essential DNA sequences) whereas multilocus deletions will be conditionally lethal at the hemizygous X-linked hprt locus. One mutagen of interest is the potent antitumor agent, mitomycin C(MMC). At lower doses, MHC induces mostly point mutations as determined by Southern blot analysis. At higher doses of MMC, a high frequency of deletion mutations is induced. These data represent the first molecular demonstration of a change in mutational spectrum with a change in mutagen dose in mammalian cells. We are currently in the process of analyzing the MMC- induced point mutations using DNA sequence analysis and we are using the pivx-recombinational recovery system to isolate deletion endpoints from genomic lambda libraries derived from selected spontaneous and MMC-induced deletion mutants. The ability to recover deletion mutants as viable clones using the AS52 cell line allows detailed studies of the mechanistic pathways by which events in the mammalian genome.
