Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.     

Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis. III. Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis

We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.     

The effect of him mutations characterized by enhanced induced mutagenesis on the spontaneous mitotic recombination in the ADE2 gene in Saccharomyces cerevisiae

We have studied the influence of him1, him2, him3 and himX mutations on the frequency of spontaneous mitotic gene conversion in the yeast Saccharomyces cerevisiae using the set of heteroallelic combinations in the ADE2 gene. Data obtained on the HIM/HIM, him/him homozygotes and HIM/him heterozygotes indicate that the him1 mutation is recessive with respect to conversion, whereas the him2, him3 and himX mutations are semidominant. Gene conversion was increased in the majority of heteroalleles of mutant diploids him1/him1. On the contrary, the him2, him3 and himX mutants have hypo-rec phenotypes on mitotic conversion. The him mutations do not affect some heteroalleles, moreover, for some heteroalleles, the effects of the him mutations was opposite. On the basis of the sum of genetical data and, particularly, of conversion event pattern in the him mutants, we suggest that him mutations analysed affect the repair pathway for mismatch correction.     

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Participation of the HIM1 gene from Saccharomyces cerevisiae in correction of heteroduplex DNA. Molecular cloning of the gene]

A group of Saccharomyces cerevisiae mutants deficient in repair of induced premutation lesions (him mutants) were previously isolated in our laboratory. Recessive him1 mutant had enhanced level of spontaneous and induced mutagenesis as well as specific altered mitotic conversion. This HIM1 gene was supposed to be involved in the process of mismatch correction of heteroduplexes. In this paper the correction efficiency of in vitro constructed heteroduplex DNA in wild-type cells and him1 mutant was studied. In the former cells heteroduplex DNA was repaired highly efficiently (about 90%), this repair efficiency being reduced in him cells approx. two times as compared with the wild-type cells. Molecular cloning of yeast chromosomal DNA fragments containing HIM1 gene was carried out. The clones were selected from the bank of yeast DNA fragments by complementing him1-1 mutation which enhances conversion frequency in ADE2 gene. One of the DNA fragments was analysed by restriction endonuclease digestion and shown to contain an insert of 6 Kb. Chromosomal integrants were obtained by homologous recombination between the plasmid and chromosomal gene him1.     

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.     

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.     

Characterization of Escherichia coli null mutants for glutaredoxin 2.

Three Escherichia coli glutaredoxins catalyze GSH-disulfide oxidoreductions, but the atypical 24-kDa glutaredoxin 2 (Grx2, grxB gene), in contrast to the 9-kDa glutaredoxin 1 (Grx1, grxA gene) and glutaredoxin 3 (Grx3, grxC gene), is not a hydrogen donor for ribonucleotide reductase. To improve the understanding of glutaredoxin function, a null mutant for grxB (grxB(-)) was constructed and combined with other mutations. Null mutants for grxB or all three glutaredoxin genes were viable in rich and minimal media with little changes in their growth properties. Expression of leaderless alkaline phosphatase showed that Grx1 and Grx2 (but not Grx3) contributed in the reduction of cytosolic protein disulfides. Moreover, Grx1 could catalyze disulfide formation in the oxidizing cytosol of combined null mutants for glutathione reductase and thioredoxin 1. grxB(-) cells were more sensitive to hydrogen peroxide and other oxidants and showed increased carbonylation of intracellular proteins, particularly in the stationary phase. Significant up-regulation of catalase activity was observed in null mutants for thioredoxin 1 and the three glutaredoxins, whereas up-regulation of glutaredoxin activity was observed in catalase-deficient strains with additional defects in the thioredoxin pathway. The expression of catalases is thus interconnected with the thioredoxin/glutaredoxin pathways in the antioxidant response.     

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.     

Mutants of Saccharomyces cerevisiae characterized by increased level of induced mutagenesis. I. Isolation and preliminary characterization of mutants

6 mutants with enhanced nitrous acid-induced reversibility of the ade2-42 allele were isolated and designated hm (high mutagenesis). Apart from sensitivity to the mutagenic exposure to nitrous acid, hm mutants were also spontaneous mutators and hypermutable under the action of UV-light and 6-N-hydroxyaminopurine. All these effects were detected not only when analysing reversibility of the ade2-42 allele, but also when scoring forward mutations in the ADE1, ADF2 genes. Gamma-mutagenesis, however, was not affected by hm mutations.     

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

Mak mutants of yeast: mapping and characterization.

Killer strains of Saccharomyces cerevisiae are those carrying a 1.5 x 10(6)-dalton double-stranded (ds) ribonucleic acid (RNA) (M) in virus-like particles and secreting a protein toxin. Most yeast (koller or not) also carry a 3 x 10(6)-dalton dsRNA (L). We have mapped mutations in eight of the chromosomal genes needed for maintaining M (mak genes). The mak genes are widely distributed on the yeast map, with no multigene complexes. We show that mutants defective in these and other mak genes lose M dsRNA, but not L dsRNA. The mak3-1 mutation results in markedly decreased cellular levels of L dsRNA, but mak3-1 stains do not lose L dsRNA completely. Mutation of mak16 results in temperature-sensitive growth, whereas mutations in mak13, mak15, mak17, mak20, mak22, and mak27 result in slow growth at any temperature. No effect of mak mutations on mating, meiosis, sporulation, germination, homothallism, or ultraviolet sensitivity has been found. The specificity of mak mutations is discussed.     

Repair of cisplatin-DNA adducts in mutants for genes controlling spontaneous and induced mutagenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast

Sensitivity to the lethal action of the anticancer substance cisplatin was studied in the yeast mutants him1, hsm2, hsm3, and hsm6, deficient for repair of spontaneous and induced mutations. The him1 and hsm3 mutants were as resistant to the agent under study as the wild-type strain. The survival of the double mutant rad2 hsm3 was higher than that of the single mutant rad2. The hsm2 and hsm6 mutants were more cisplatin-sensitive than the wild type. Cisplatin was shown to have high mutagenic and recombinogenic effects on yeast cells.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.     

Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme.     

Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model.

Direct selection for 12 mutations (blm) conferring hypersensitivities to lethal effects of bleomycins in Saccharomyces cerevisiae resulted in mutants exhibiting cross-hypersensitivity to ionizing radiation and hydrogen peroxide. Remaining mutations did not confer cross-hypersensitivity to radiation. All blm mutations were recessive, except codominant blm3-1, and were assigned to seven complementation groups.     

Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus

Summary Twenty-four sporeless mutants were isolated from an Amut Bmut strain (mutant in the incompatibility factors) of the basidiomycete Coprinus cinereus. All the sporeless mutations were recessive to the wild type. These mutants and a previously isolated recessive sporeless strain, N2-7 (Kanda and Ishikawa 1986) were crossed with a wildtype strain. An F1 random spore analysis indicated that sporulation deficiencies in these mutants were caused by single nuclear gene mutations. These mutations were all complementary to each other, thus twenty-five sporulation genes were identified. Five of them were linked to the A incompatibility factor. Cytological observations classified these mutants into the following four types according to the stage of the blockage: (1) meiosis stopped at meta-anaphase I; (2) meiosis was completed, but further basidial development did not occur; (3) basidial development stopped at the sterigma stage; (4) basidial development stopped at the prespore stage.