MOLECULAR STRUCTURAL-ANALYSIS OF 417 HPRT MUTATIONS INDUCED BY RESTRICTION ENDONUCLEASES IN CHINESE-HAMSTER OVARY (CHO) CELLS

CHO cells were exposed to 11 different restriction endonucleases by el ectroporation and their mutagenicity was measured. Nine of them have o ne or more recognition sites within exons of the HPRT gene, whereas th e remaining two cut in introns only. The mutagenic efficiency of the v arious enzymes varied markedly; mutagenicity of Sau3AI was considerabl y higher than that of the other enzymes. Neither cytotoxicity nor muta genicity could be related to the number or location of recognition sit es within the cDNA. A total of 417 independent restriction enzyme indu ced mutant clones were isolated from 20 separate experiments for molec ular analysis; all nine exons of the HPRT gene were analyzed by a modi fied multiplex deletion screening method with polymerase chain reactio n (PCR) amplification. Among spontaneously arising mutants, 70.8% show ed no change in PCR pattern, indicating a small scale change (point mu tation), whereas partial deletions were observed in 24.7%, and total d eletions in 4.5% of mutant clones. In contrast, approximately 70% of r estriction enzyme induced mutants showed partial or total deletions. T here was no obvious relationship between type of break (blunt versus s taggered ends), and the DNA structure of the mutations induced. For pa rtial deletions, the distribution of breakpoints within introns appear ed to occur at random, and did not correlate with the mutagenicity of a given enzyme. Thus, though DNA double-strand breaks appear to be imp ortant mutagenic lesions that can induce a high frequency of deletion mutants, no specific relationship of mutagenic potential to the type o f breaks, their sites within the HPRT gene or the molecular structure of the mutations induced could be identified.