We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in fi-galactosidase. Each strain is Lac-and reverts to Lac' only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac-to Lac' frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains.One of the first steps toward elucidating mutagenic pathways is to determine the specificity of mutations generated spontaneously or by treatment with mutagens. In many cases, identifying the type of mutations produced yields important information on the nature of the premutagenic lesion, as well as the role of cellular systems in processing damaged DNA (reviewed in ref. 1). Toward this end, systems have been developed that allow the rapid determination of mutagenic specificity by genetic methods. For instance, reversion of trpA alleles was utilized by Yanofsky and coworkers (2) to examine mutagenic specificity in Escherichia coli. In yeast, Sherman and coworkers (3, 4) utilized the reversion of characterized mutations in the isocytochrome c system to deduce the base substitution specificity of different mutagens. Ames and colleagues (5,6) monitored the reversion of specific mutations in the his operon of Salmonella typhimurium to allow the rapid screening of base substitution and frameshift activity of a large number of mutagens. This work was important because it established a strong correlation between carcinogens and mutagens (6). Levin and Ames (7) have modified this system to allow the determination of specific base substitutions, utilizing the reversion of nonsense codons, followed by analysis of the revertants, a principle first used by Person and coworkers (8). The lad system (9, 10) provides a more detailed look at mutagenic specificity by examining the forward spectrum of nonsense mutations produced in the lacI gene of E. coli. Mutations at >80 sites can be monitored. In addition to these genetic methods, the direct DNA sequence determination (11)(12)(13)(14)(15)(16) and colony hybridization with specific probes (17) have also been used for the determination of the nature of spontaneous and induced mutations.All of the above systems require further analysis to determine the ability of a mutagen to produce each of the six possible base substitutions. A system that allows the determination of each base substitution, simply by measuring the number of revertant colonies that appear on a plate, would great...

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of g3-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G-C --TA transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the IacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in ANT -C-G transversions.Mutants with higher than normal rates of spontaneous mutation have facilitated our understanding of mutational pathways. Some of the "mutator" strains have characterized defects in postreplication mismatch repair (1, 2), in specific glycosylases (3), or in the editing function provided by the e subunit of DNA polymerase III (4). Characterization of additional mutators may reveal pathways of mutagenesis and repair. Toward this end, we have used a highly sensitive screening method to detect mutator strains that revert defined mutations in lacZ by a limited number of base substitutions. Here we report the characterization of a mutator locus, mutY, which results in the specific generation of G-C

Background & Aims-The human mutyh gene encodes a base excision repair (BER) protein that prevents G:C to T:A transversions in DNA. Biallelic mutations in this gene are associated with recessively inherited familial colorectal cancer. The aim of this study was to characterize the functional activity of mutant-MUTYH and SNP-MUTYH proteins involving familial colorectal cancer.

The macronucleus of Tetrahymena thermophila contains a single actin gene. We have isolated this gene from a partial plasmid library by using the yeast actin gene as a probe. The nucleotide sequence of the gene has been determined and the amino acid sequence of the potential protein deduced. The encoded protein is 375 amino acids long, one amino acid longer than the yeast actin. It is one of the most divergent actins sequenced to date, being only 75% homologous to yeast actin. Unlike the actin genes from most other organisms, it does not contain introns. The coding region contains TAA and TAG codons; the translation termination codon is TGA. Comparison of the amino acid sequence of the Tetrahymena actin with that of actins from other organisms suggests that TAG may code for glutamic acid. The gene is transcribed from multiple initiation sites between 57 and 98 nucleotides upstream of the translation start codon. The 5' flanking region is very A+T-rich and contains numerous "TATA-like" sequences upstream of the transcription start sites.

The Vsr endonuclease of Escherichia coli initiates the repair of T/G mismatches caused by deamination of 5-methylcytosine to thymine. In this paper, we examine the capacity of Vsr to prevent CG-to-TA mutations in cells with increased transcription of the cytosine methylase gene (dcm). We find that sufficient Vsr is produced by a single chromosomal copy of vsr to prevent mutagenesis. We also investigate the cause of the transition and frameshift mutations in cells overproducing Vsr. Neither the absence of the dcm methylase nor its overproduction affects Vsr-stimulated mutagenesis. However, addition of mutS, mutL, or mutH on multicopy plasmids has a significant effect: mutL or mutH decreases the number of mutations, while mutS stimulates mutagenesis. The mut-containing plasmids have the same effect in cells treated with 2-aminopurine and in cells made defective in DNA proofreading, two experimental situations known to cause transition and frameshift mutations by saturating mismatch repair.The Vsr endonuclease in Escherichia coli cleaves DNA at T/G mismatches in C(T/G)WGG and related sequences (W ϭ A or T), producing a single-strand nick 5Ј of the T. This is the first step in very short patch (VSP) repair, the end result of which is the replacement of the mismatch with a normal CG base pair (reviewed in reference 17). Since the dcm cytosine methylase of E. coli methylates the second C of the sequence CCWGG, the main function of VSP repair is probably the prevention of CG-to-TA mutations caused by deamination of 5-methylcytosine to thymine. Cells which contain dcm but not vsr have high frequencies of C-to-T mutations at 5-methylcytosine (16,20).The chromosomal arrangement of dcm and vsr is unusual: the 5Ј end of vsr overlaps the 3Ј end of dcm in a ϩ1 reading frame (25). The two genes are probably transcribed as a single mRNA, with translation of vsr depending on translation of dcm (7). The arrangement of the genes suggests that coordinated production of the two proteins is important. For example, it could ensure that Vsr is present whenever the methylase is present, thereby minimizing mutations caused by 5-methylcytosine deamination.Recently, we uncoupled the expression of vsr from dcm by cloning vsr into a multicopy plasmid under the control of the strong, constitutive trc promoter (8). We had hypothesized that elevated levels of Vsr in the cell would increase competition between VSP repair and postreplication mismatch repair (MMR) for the correction of T/G mismatches formed by DNA polymerase errors and that this would result in increased numbers of CTAGG-to-CCAGG mutations. As predicted, these mutations were elevated. Surprisingly, however, overproduction of Vsr also increases transition mutations in general and dramatically stimulates frameshifts. Thus, the unusual genetic arrangement of dcm and vsr may also serve to minimize mutations caused by Vsr. For example, the overlap between the coding regions of the two genes may decrease the efficiency of vsr translation.One purpose of the current study was to further i...