Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells

Abstract

To determine the types of gene structural alterations causing deficiency of adenine phosphoribosyl transferase (aprt) activity in spontaneous and chemically induced mutations of cultured somatic cells, we analyzed the restriction enzyme cleavage patterns of aprt gene sequences in mutant strains selected from Chinese hamster ovary cells. Patterns of aprt-containing fragments in Southern blots were mostly unchanged in our collection of 280 ethyl methane sulfonate-induced and spontaneous aprt- mutants, suggesting that base-pair changes or other alterations below our limit of resolution on agarose gels (approximately 50 base-pairs) are responsible for the great majority of mutations at the aprt locus. Occasionally, these mutations could be localized when they resulted in the loss or gain of a restriction enzyme site and the generation of new fragments of predictable size. Deletions of aprt-containing sequences were detected in only eight of 119 spontaneous mutants and in only one ethyl methane sulfonate-induced mutant. An insertion of 300 base-pairs near the 5' end of the aprt structural gene was found in one spontaneous aprt- strain. This insertion mutant was stable with a reversion frequency of less than 2 X 10(-7). Several unstable aprt- mutants were detected in our collection, but these had no observable alterations of aprt coding or flanking sequences.