MOLECULAR ANALYSIS OF DELETION MUTATIONS IN CHINESE HAMSTER OVARY CELLS

点击翻译按钮获取中文摘要

The Chinese hamster ovary (CHO) cell line, AS52, carries a single functional copy of the bacterial gpt gene stably integrated into the CHO genome. Mutations at gpt are recovered as 6-thioguanine resistant colonies and may arise in these cells as a result of the loss of functional gpt sequences through i) intrachromosomal deletion, ii) mitotic recombination or iii) gene conversion. The site of integration of the gpt locus appears to allow the recovery these complex mutations where such events will be conditionally lethal at the analogous hemizygous X-linked hprt locus. Thus, AS52 cells are sensitive to induced mutagenesis by a variety of clastogens which are often classified as nonmutagens in other assays. Two agents under study are 5-azacytidine (5AC) and 1-beta-D-arabino-furanosylcytosine (ara-C). Neither 5AC nor ara-C are mutagenic at the hprt locus but both are potent mutagens at the gpt locus in AS52 cells. Ara-C has been demonstrated to generate deletions or recombination/gene conversion events and efforts are underway to define the spectrum of mutations induced by 5AC. We are continuing to refine the AS52 cell system to provide a more precise description of complex mutations. Efforts are underway to evaluate the requirement for perfect sequence homology and/or a role for mismatch repair in the regulation of mitotic recombination in mammalian cells. Similar studies in prokaryotes and lower eukaryotes have implicated mismatch repair in the regulation of recombination. These studies have direct bearing on the mechanisms by which mutations are induced by agents that saturate or inhibit mismatch repair. Such agents may be particularly recombinogenic and the AS52 cell line may be uniquely capable of detecting these events. Finally, we are continuing to use the pi vx-system to isolate deletion endpoints for molecular characterization from lambda genomic libraries derived from selected AS52 mutants.