ANALYSIS OF 2ND-STEP MUTATIONS OF CLASS-II AND CLASS-III CHO APRT HETEROZYGOTES - CHROMOSOMAL DIFFERENCES IN DELETION FREQUENCIES

We have determined the nucleotide sequence surrounding a BclI restriction fragment length variation near the aprt gene of CHO cells. By BclI digestion of the PCR-amplified DNA from a variety of APRT-deficient mutants derived from CHO cells, we were able to infer the following. First, all three heterozygotes of class II, which are know to undergo the second mutational step via a large deletion event occurring at high frequency, are mutant at the chromosome Z4-linked allele, and wild type at the Z7 allele. Second, both class-III heterozygotes, which mutate to the APRT- phenotype at low frequency, are mutant at the Z7 allele, wild type at the Z4 allele. A total of 12 class-I lines, defined as having already undergone a deletion event and yielding fully APRT- mutants at low frequency were all found to have lost the Z7-linked allele. We conclude that the Z7-linked allele is substantially more susceptible to mutation by the large deletion event than is the Z4-linked allele. This supports a hypothesis we advanced earlier to explain the existence of the class-III heterozygotes but does not support previous work suggesting that a chromosomal inversion break-point junction near the Z4-linked aprt allele is responsible for the high frequency deletion event.