Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Self Cite

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 coligpt gene, coding for the enzyme xanthine (guanine) phosphoribosyltransferase (GPT; EC 2.4.2.22). We had 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.

Section: Methodsmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 95%

A sensitive molecular assay for mutagenesis in mammalian cells: reversion analysis in cells with a mutant shuttle vector gene integrated into chromosomal DNA.

Gelbert¹,

Davidson²

1988

Self Cite

“…The DNA sequences associated with the possible GPTI revertants of each cell line are also presented in Table 1. Revertant lines possessing each of the possible revertant DNA sequences presented have been isolated in previous studies from this laboratory (22,32,33). For lines B12 and B20, reversion has been observed to occur only by a G'C -* A-T transition at the site of the original mutation and restoring the wild-type DNA sequence.…”

Section: Resultsmentioning

confidence: 99%

“…The latter type reversion events result in nonwild-type revertant DNA sequences. Thus, based on the types of reversion events that have been observed with lines S1, S44, B12, and B20, the sequences associated with these lines make it possible to use reversion analysis to assess thymidine mutagenesis at the 5' and 3' guanine residues of a Mutant sequences and all observed revertant sequences (21,32,33) GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet.…”

Section: Resultsmentioning

confidence: 99%

Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo.

Kresnak

Davidson

1992

Self Cite

Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyltransferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency ofmutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G-C -A-T transition mutations that occur preferentially at the 3' ganine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo.The fidelity of DNA replication in mammalian cells strongly depends upon a balanced supply of deoxyribonucleotide triphosphates available as precursors for DNA synthesis. Mutagenesis as a result of nucleotide pool perturbations has been examined in a number of eukaryotic (1-5) and prokaryotic (6, 7) systems. In cultured mammalian cells, alteration of the intracellular precursor pools can result in mutations (8,9) and in gross chromosomal abnormalities, such as large deletions (10), breaks (11), and sister chromatid exchanges (12). Thymidylate deprivation induces mutation in bacteriophage T4 (13), mitotic recombination in yeast (14), and the RecAdependent error-prone response in Escherichia coli (15). Precursor pool imbalances also disrupt the accuracy of DNA synthesis in cell-free DNA replication systems (16, 17).In cultured mammalian cells, perturbation of the intracellular nucleotide precursor pools can be accomplished by exposing the cells to high concentrations of exogenous deoxyribonucleosides or their analogs. One commonly used agent is the thymidine analog 5-bromodeoxyuridine (BrdUrd). Apparently, high levels of exogenous BrdUrd disrupt intracellular deoxycytidine metabolism (through inhibition of the enzyme ribonucleotide reductase) such that the dCTP pools available for DNA synthesis are reduced (18)(19)(20), resulting in misincorporation of BrdUrd opposite guanine residues. Experiments involving mutagenesis of a bacterial gp...

“…The frequency of a particular base change that restores a wild-type DNA sequence can be measured by the frequency of phenotypic revertants, permitting the collection of large amounts of data about molecular events in the absence of marker recovery and DNA sequencing. An assay measuring phenotypic reversion of a bacterial transgene within mouse cells has been described, but determination of the molecular basis of reversion entailed both transgene recovery and sequencing, since second-site reversion was observed and no readily assayed markers for molecular events were described (8,25). Since this report describes mutation at a specific nucleotide pair, one cannot generalize from these data about the frequency at which the same mutations might occur at other sites.…”

mentioning

confidence: 99%

Mouse transgenes in human cells detect specific base substitutions.

Schaff¹,

Jarrett²,

Dlouhy³

et al. 1990

We describe a system of transgenic human cell lines that detects and identifies specific point mutations at defined positions within a gene. The target transgenome is a mouse adenine phosphoribosyltransferase (APRT) gene rendered nonfunctional by introduction of a substitution at either of two bases that comprise a splice acceptor site. Reversion at a mutated site results in the expression of wild-type mouse APRT and consequent growth of APRT' transgenic cell colonies. Site-specific reversion to wild-type sequence is confirmed by regeneration of a previously destroyed diagnostic Pst I site. Two independent cell clones, each with mutant transgenomes bearing an A -* G transition, exhibited an up to 7500-fold, dose-dependent induction of reversion following treatment with ethyl methanesulfonate. Treatment of these clones with 2-aminopurine resulted in no induction of revertants. In contrast, another transgenic cell clone, bearing a G -b A transition, reverted as a consequence of 2-aminopurine, but not ethyl methanesulfonate, treatment. These data confirm for human cells the proposed mechanisms of action of these mutagens and provide evidence for the utility of our site-specific reversion method for mutagenesis studies.There are no mammalian cell assays that precisely define, without the aid of DNA sequencing, the type(s) of mutation that an exogenous agent can produce. In contrast to the Ames Salmonella test (1-3), most mammalian cell mutagenesis assays measure forward mutation at a target locus whose altered phenotype is drug resistance (4). Although useful for detecting agents that cause mutations, such assays provide little information regarding the type(s) of mutation produced, unless the nucleotide sequence of the mutant target gene is determined in each case (5, 6). To circumvent the necessity for DNA sequencing (e.g., refs. 7 and 8), we have developed a detection system that relies on site-and sequence-specific reverse mutation. First, a defined point mutation is introduced into a wild-type mouse adenine phosphoribosyltransferase (APRT) gene. Then, the mutated gene is transfected into a human recipient cell with a stable APRT-phenotype. Only reverse mutation to the wild-type mouse gene sequence is detected by expression of the selectable mouse APRT+ phenotype and growth of cell colonies, thereby defining the precise mutation that has occurred. The site within the target gene at which mutations have been introduced was chosen so that second-site mutations would be unlikely to produce phenotypic reversion, and none have been detected.The mouse APRT gene was chosen as the target transgenome primarily because the expression or absence of the encoded enzyme provides the basis for sensitive forward and backward chemical selection of cultured cells (9). This mammalian gene is also small [-3 kilobases (kb)], which facilitates its in vitro manipulation; its nucleotide sequence and its structural organization are known (10); and its encoded product (mouse APRT) is electrophoretically distinguishable from the h...