We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase (XGPRT) gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) CHO cells which have been transformed by the plasmid vector pSV2gpt (10, 11). pSV2gpt carries the Escherichia coli gpt gene and through the use of the simian virus 40 early promoter expresses the bacterial gene for XGPRT in mammalian cells. One isolated CHO transformant, designated AS52, carries a single copy of the gpt gene stably integrated into the high-molecularweight DNA. Bacterial XGPRT and mammalian HGPRT are functionally analogous except that under physiological conditions XGPRT catalyzes the formation of xanthine 5'-monophosphate from xanthine, whereas HGPRT does not. Selection protocols utilizing the purine analog 6-thioguanine for the study of mutation at the hgprt locus in CHO cells (12) can be used to select for mutations at the single gpt gene in the pSV2gpt-transformed CHO line. In this study, we show that mutation induction at the gpt locus in pSV2gpt-transformed CHO cells can be quantified and that specific deletion mutations which affect gpt gene function are the basis for the observed 6-thioguanine-resistant (Tg') phenotype in two mutants derived from this transformed line.The parental line, CHO-K1-BH4 (hgprt+), has been previously described (6). The X3/5 line (HGPRT-), which served as the transformation host for the pSV2gpt gene transfer experiments, was derived from the CHO-K1-BH4 line after an X-ray irradiation of 500 rads and selection in 10 ,uM 6-thioguanine; several lines of evidence indicate that it carries a stable, nonrevertible mutation at the hgprt locus (R. Machanoff, M.S. thesis, University of Tennessee, Knoxville, Tenn., 1982 supplemented with 5% heat-inactivated and dialyzed fetal bovine serum (F12FCM5) at 5% C02, 37°C, and 100%o humidity.The AS52 line is a gpt transformant of X3/5 which carries a single ...

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The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.

The phenotypic expression time of ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants was studied with Chinese hamster ovary cells in culture (CHO/HGPRT system). After mutagen treatment of exponential phase cultures, the cells were maintained either in the exponential phase through subculture in medium containing 5% dialyzed fetal bovine serum (FBS) or in a nondividing viable state by use of medium containing 0-1% dialyzed FBS. The time course of expression of the 6-thioguanine-resistant phenotype was similar with both exponential phase and division-arrested cultures showing maximum expression by 9 days after mutagen treatment, and both methods of expression also yielded similar mutant frequencies over a range of EMS concentrations. This study shows that once the mutagenic event is fixed, the expression of the mutant phenotype does not require continued cell division since it occurs in division-arrested cultures. These results also suggest that both dilution of pre-existing hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme by cell division and turnover by protein degradation are involved in the phenotypic expression. Both processes occur in exponential cultures, but only protein turnover in arrested cultures. Consistent with this was the demonstration that the rates of total cell protein turnover increased in division-arrested cultures maintained in serum-free medium. These results separate genetic damage and phenotypic expression in a temporal sense, and point out the need to consider the mechanisms responsible for each process involved in the induction and expression of mutations.

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