Detection of the DNA primary structure modifications induced by the base analog 6-n-hydroxylaminopurine in the alpha-test in yeast saccharomyces cerevisiae (original) (raw)
Related papers
Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae
Biochemical and Biophysical Research Communications, 2004
To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 • 10 À7 , 100-fold lower than that in diploid cells (1.03 • 10 À4). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C fi A:T and G:C fi T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.
Yeast, 1996
A strategy for gene replacement in Succhuromyces cerevisiae has been modified to facilitate the repeated substitution of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the desired mutation and is free of extraneous vector DNA. The construction of an internally deleted chromosomal target locus carrying the counterselectable CYH2' marker and a second positively selectable marker has been simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained upon the subsequent integration of in vitro mutated DNA. The modified chromosomal target locus is amenable to replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (>70%); (ii) single-step integration of a linear segment of mutated genomic DNA by selection for cycloheximide resistance. A subsequent screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency (22%). Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring these variously modified chromosomal loci.
Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene
Proceedings of the National Academy of Sciences, 1999
Heterozygosity for germ-line mutations in the DNA mismatch repair gene MSH2 predisposes humans to cancer. Here we use a highly sensitive reporter to describe a spontaneous mutator phenotype in diploid yeast cells containing a deletion of only one MSH2 allele. We also identify five MSH2 missense mutations that have dominant mutator effects in heterozygous cells when expressed at normal levels from the natural MSH2 promoter. For example, a 230-fold mutator effect is observed in an MSH2͞msh2 diploid strain in which Gly 693 , which is invariant in MutS homologs and involved in ATP hydrolysis, is changed to alanine. DNA binding data suggest that mismatch repair is suppressed by binding of a mutant Msh2-Msh6 heterodimer to a mismatch with subsequent inability to dissociate from the mismatch in the presence of ATP. A dominant mutator effect also is observed in yeast when Gly 693 is changed to serine. An early onset colorectal tumor is heterozygous for the analogous Gly 3 Ser mutation in hMSH2, and a second hMSH2 mutation was not found, suggesting that this missense mutation may predispose to cancer via a dominant mutator effect. The mutator effects of the deletion mutant and the Gly 3 Ala missense mutant in yeast MSH2 are enhanced by heterozygosity for a missense mutation in DNA polymerase ␦ that reduces its proofreading activity but is not a mutator in the heterozygous state. The synergistic effects of heterozygosity for mutations in two different genes that act in series to correct replication errors may be relevant to cancer predisposition.
Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology
2003
This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.
Proceedings of the National Academy of Sciences, 1983
Several cis-acting mutations that prevent homothallic mating type conversions in Saccharomyces cerevisie have been examined. Deletions within the mating type (MAT) locus were obtained by selecting for survivors among homothallic MATa cells carrying the rad52 mutation. The survivors were unable to switch mating type, even in RADW derivatives. The deletions varied in size from fewer than 50 to more than 750 base pairs. All of the deletions removed a Hha I site at the border between the a-specific sequences (Ya) and the adjacent Z region. We also examined several spontaneous inc mutations that prevent MAT switching. Two of these mutations were cloned in recombinant DNA plasmids and their sequences were determined. The MATa-inc 3-7 mutation proved to have an altered Hha I site at the Ya/Z border, by virtue of a single base pair substitution G'C --A-T in the second base pair of the Z region (Z2). Restriction fragment analysis showed that two other independently isolated strains with MATainc mutations had altered the same Hha I site. The MATa-inc 4-28 mutation contains a single base pair substitution COG -+ TA at position Z6. A base pair difference at position Zn1 in two MATa strains does not affect MATa conversions. We conclude that the region near the Y/Z border is essential for the efficient switching of MAT alleles and constitutes an enzyme recognition site for a specific nucleolytic cleavage of MAT DNA.
MGG Molecular & General Genetics, 1994
To study chemically induced DNA amplifications we used the haploid Saccharomyces cerevisiae strain TR(MS1)-I carrying an integrated chromosomal copy of the human minisatellite, MS1. Chemicals with different mechanisms of action were tested in this strain: methyl methanesulphonate, ethylene oxide (EO), propylene oxide (PO), camptothecin, 2,3,7,8-tetrachlorodibenso-pdioxin (TCDD) and reserpine. No increase in frequency of new MS1 length alleles was seen with any of the tested chemicals relative to the spontaneous frequency of approximately 30%. EO and TCDD induced changes in the amplification spectrum, i.e. the frequency distribution of MS1 length alleles longer than the original 1.42 kb allele. PO and camptothecin increased the frequency of plasmid "pop-out" events. It seems likely that several mechanisms e.g. unequal exchanges, replication slippage and loop formation leading to deletion of a ring of tandem repeats, are involved in the generation of new MS1 length alleles. A loop-forming deletion mechanism is supported by the tendency to multimodality shown in the deamplification (loss of repeat units) spectra, i.e. the frequency distribution of new MS 1 length alleles shorter than the original allele. EO and TCDD induced "longer" MS1 length alleles as compared to the control. The frequent generation of new MS 1 length alleles in this haploid yeast strain further demonstrates the instability of such sequences and their possible relevance to genetic toxicology and the mechanisms of induction of cancer as well as other diseases. This study is a first step towards the development of an assay for DNA amplification without the use of a selective agent.
Russian Journal of Genetics, 2009
We studied the effect of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP)de novo and purine salvage and interconversion pathways, on sensitivity of yeast Saccharomyces cerevisiae to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine(AHA). It was shown that the manifestation of HAP and AHA mutagenic properties depends on the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of any step of IMP biosynthesis, with the exception of the block mediated by inactivation of genes ADE16 and ADE17 leading to the accumulation of 5-aminoimidazole-4carboxamide ribonucleotide (AICAR), was shown to enhanceyeast cell sensitivity to the HAP mutagenic effect; however, it does not affect the sensitivity to AHA. A block of conversion of IMP into adenosine monophosphate (AMP) causes hypersensitivity of yeast cells to the mutagenic action of HAP and to the toxic effect of HAP, AHA, and hypoxanthine. It is possible that this enhancement of sensitivity to HAP and AHA is due to changes in the pool of purines. We conclude that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine salvage and interconversion in yeast, make the greatest contribution to the protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are discussed.
Molecular and cellular biology, 1991
We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also create...
Use of a reporter gene assay in yeast for genetic analysis of DNA-protein interactions
Methods in molecular biology (Clifton, N.J.), 2009
We describe methods for the genetic analysis of a DNA-protein interaction from any species. The DNA-binding domain of the protein of interest is expressed in yeast cells as a fusion with a known transcriptional activation domain, and the target binding site is used as an artificial upstream activation sequence (UAS) in an engineered promoter driving expression of a reporter gene, such as beta-galactosidase. Expression of the reporter gene is dependent upon specific, high-affinity interaction between the DNA-binding domain of the artificial activator and the synthetic UAS. Error-prone PCR is used to introduce mutations into either member of this interacting pair, and homologous recombination is used to return the mutagenized sequences to their proper sequence contexts in vivo. Altered expression of the reporter gene is then used as a screen or selection for mutations conferring the desired phenotype, such as reductions or increases in the stability of the DNA-protein complex. Followi...