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For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10-20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.

We have analyzed the specificity of mutations induced by ethyl methanesulfonate (EtMes) in mouse cells carrying a selectable bacterial gene. The target gene was the Escherichia coli gpt gene contained within a retroviral shuttle vector integrated into mouse chromosomal DNA. Following mutagenesis by EtMes, cells with mutations in the gpt gene were selected as resistant to 6-thioguanine. Shuttle vector sequences were recovered from the mutant cell lines following fusion with monkey COS cells and introduced into bacteria as part of a bacterial plasmid. The DNA base sequences of the mutant genes were directly determined from plasmid DNA. All of the EtMes-induced mutations involving single base changes were found to be G:C to A:T transitions.

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.

A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells. Plasmid extracted from the transformed mouse cells was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. Approximate mutant frequencies of 3-16 X 10(-3) were observed for plasmid molecules which had been passaged through the mammalian cells. Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures.

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate (dNTP) pools were examined in several Syrian hamster melanoma cell mutants which are resistant to hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and which also show increased resistance to bromdeoxyuridine (BrdU) mutagenesis. For most of the mutants, resistance to HU and BrdU mutagenesis is associated with increased levels of ribonucleotide reductase activity. No evidence was found for qualitative alterations in the ribonucleotide reductase in the mutant cells. The dNTP pools in the mutants are somewhat resistant to the perturbations can be produced in wild-type cells by the addition of BrdU, although significant perturbations can be produced in the mutants by higher concentrations of BrdU. The decrease in BrdU-induced nucleotide pool perturbations may account for the resistance of the mutants to BrdU mutagenesis.

Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells.

We have developed a system for the molecular analysis of mutations in mammalian cells. This system is based upon the use of mammalian cell lines containing mutant shuttle vector genes integrated into chromosomal DNA. The target for mutation was the Escherichia coli gpt gene, coding for the enzyme xanthine (guanine) phosphoribosyltransferase (GPT; EC 2.4.2.22). We have previously isolated a large number of cell lines containing mutant gpt genes with single base changes. From these lines, revertants were selected on the basis of the reappearance of GPT activity. In general, the frequency of revertants was below 10(-7). The gpt genes were recovered from 32 revertants and sequenced to determine the nature of the base changes associated with reversion. In the majority of the revertants, there was a base change within the originally mutated codon, leading to either restoration of the wild-type amino acid sequence or substitution of a different amino acid at the original mutated site. In no case did reversion of a base substitution mutant involve an amino acid residue other than that affected by the original mutation. The results have demonstrated a number of sites in the GPT polypeptide at which amino acid substitutions are compatible with enzyme activity and one site at which the loss of an amino acid is compatible with enzyme activity. This study establishes reversion analysis as a sensitive molecular assay for mutagenesis in mammalian cells.

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.

We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 of the 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base-pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. All 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.

When cells of a Syrian hamster melanoma were grown in increasing concentrations of bromodeoxyuridine (BrdU), lines of mutant cells able to grow well at high concentrations of BrdU were isolated. The mutant cells were characterized by BrdU dependence. In the absence of BrdU, the cells grew very poorly. The requirement for BrdU was specific for both the bromine and the deoxy sugar. The mutant cells incorporated BrdU into the DNA, replacing about 50% of the thymine residues with bromouracil. The reason for the BrdU dependence is not known.