Nature of mutations conferring resistance to 8-azaguanine in mouse cell lines.

Two stable mouse cell mutants Ao and RAG, which are resistant to 8-azaguanine and deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT), have been studied in order to establish the nature of molecular changes conferring the mutant phenotypes. A specific precipitating rabbit antiserum was prepared against the normal HGPRT purified from mouse tissues, and used to test for cross-reacting material (CRM) in the mutant lysates. Neither mutants contained detectable cross-reacting material, as demonstrated by precipitation inhibition tests. However, the L cell-derived mutant A9 was shown to have a low but significant level of HGPRT activity which was clearly different from that of the normal enzyme in the wildtype parental cell line. Compared to the wild-type enzyme, the HGPRT in Ao is extremely heat labile, and has an elevated substrate-binding constant in addition to distinct antigenic differences. Both A9 and RAG have been shown previously to revert to the normal phenotype with low frequency, thus ruling out gene deletions as a possible cause of the 8-azaguanine resistance. It is suggested that RAG could involve a recessive regulatory mutation, while A9 may contain a structurally altered HGPRT as a result of a missense mutation within the structural gene for this enzyme.