The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC--AT transitions or AT-*TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT-*GC transitions and for one cell line with a GC-*CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT--GC transitions had mutated back to the wild-type gpt sequence via GC-*AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC->CG transversion had mutated via GC--AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG--GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.Analysis of the molecular mechanisms of mutagenesis in mammalian cells has been hindered by the inability to efficiently recover chromosomal genes from the cells. To overcome this problem, our laboratory recently has developed a system (3) that combines mutagenesis of a gene that is chromosomally integrated in mammalian cells, clonal purification of the mutant cells, excision of the gene, and transfer of the gene into bacteria for DNA sequencing. The key feature of this system is the use of a retroviral shuttle vector as the element in which the gene is located. The target gene for mutagenesis is the Escherichia coli gpt gene, which codes for the enzyme xanthine-guanine phosphoribosyltransferase (GPT). The gene was incorporated into the retroviral vector pZipNeoSV(X)1 (5) and introduced into murine A9 cells, which lack activity of the enzyme hypoxanthine-guanine phosphoribosyltransferase (10), a mammalian enzyme that is similar in function to GPIT. In hypoxanthineguanine phosphoribosyltransferase-deficient cells, transformants that produce functional GPIT can be selected in HAT medium, and those that have lost the ability to produce functional GPIT can be selected with 6-thioguanine. The retroviral vector has long terminal repeats, which allow it to integrate into mam...

The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied. Murine cells with single mutant copies of the E. coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected. Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted. All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions. The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis. Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol. The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region. However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis. For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis. Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.