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  • 1
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mutagen hyperresistance ; Southern, Northern analysis ; Gene transplacement ; Transposon mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The genes SNQ and SFA confer hyperresistance to 4-NQO and FA when present on a multi-copy plasmid in yeast. Both are non-essential genes since transplacement of SNQ by a disrupted snq-0::LEU2 yielded stable and viable haploid integrants. Southern analysis revealed that SNQ and SFA are single-loci genes, and OFAGE analysis showed that they are located on chromosome XIII and IV, respectively. Northern blot analysis of SNQ and SFA revealed poly(A)+ RNA transcripts of 2 kb and 1.7 kb, respectively. Nuclease S 1 mapping showed SNQ to have a coding region of 1.6 kb and SFA, one of 1.3 kb. The 5′ coding regions were determined for both genes, while the 3′ end could only be determined for gene SNQ. Both genes do not appear to contain introns. The SFA locus was also mapped by transposon mutagenesis. Tn10-LUK integrants disrupted the SFA gene function at sites that were determined by subcloning to lie within the SFA transcription unit.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0983
    Keywords: Mutagen hyper-resistance ; Nitrogen mustard ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementations group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 11 (1986), S. 211-215 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Hyperresistance ; DNA damaging agents ; Genotoxic effects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinohne-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0983
    Keywords: Formaldehyde ; DNA-protein cross-links ; Repair ; Saccharomyces cerevisiae ; Hyperresistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The formation and removal of formaldehyde-mediated DNA protein cross-linking was measured by CsCI density gradient analysis in yeast strains of differing resistance to formaldehyde. Wild-type cells and transformants made hyperresistant to formaldehyde by a multi-copy vector containing the yeast SFA gene were specifically labeled in their DNA and incubated in the presence of formaldehyde. Treatment with formaldehyde lead to the formation of equal amounts of DNA protein cross-links; subsequent liquid holding of cells for 24 h resulted in the removal of nearly all DNA protein crosslinks regardless of the original formaldehyde resistance status of the strains.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Multiple mutants of DNA repair ; Sensitivity to nitrogen mustard and to radiation ; Thermoconditional DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 ts were found epistatic to genes of the rad3 group, whereas snm2-1 ts was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-0983
    Keywords: Key words Psoralen sensitivity ; Cytochrome oxidase ; Saccharomyces cerevisiae ; Oxidative stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast gene PSO7 was cloned from a genomic library by complementation of the pso7-1 mutant's sensitivity phenotype to 4-nitroquinoline-1-oxide (4NQO). Sequence analysis revealed that PSO7 is allelic to the 1.1-kb ORF of the yeast gene COX11 which is located on chromosome XVI and encodes a protein of 28-kDa localized in the inner mitochondrial membrane. Allelism of PSO7/COX11 was verified by non-complementation of 4NQO-sensitivity in diploids homo- and hetero-allelic for the pso7-1 and cox11::TRP1 mutant alleles. Sensitivity to 4NQO was the same in exponentially growing cells of the pso7-1 mutant and the cox11::TRP1 disruptant. Allelism of COX11 and PSO7 indicates that the pso7 mutant's sensitivity to photoactivated 3-carbethoxypsoralen and to 4NQO is not caused by defective DNA repair, but rather is due to an altered metabolism of the pro-mutagen 4NQO in the absence of cytochrome oxidase (Cox) in pso7-1/cox11::TRP1 mutants/disruptants. Lack of Cox might also lead to a higher reactivity of the active oxygen species produced by photoactivated 3-carbethoxypsoralen. The metabolic state of the cells is important for their sensitivity phenotype since the largest enhancement of sensitivity to 4NQO between wild-type (WT) and the pso7 mutant occurs in exponentially growing cells, while cells in stationary phase or growing cells in phosphate buffer have the same 4NQO resistance, irrespective of their WT/mutant status. Strains containing the pso7-1 or cox11::TRP1 mutant allele were also sensitive to the oxidative stress-generating agents H2O2 and paraquat. Mutant pso7-1, as well as disruptant cox11::TRP1, harboured mitochondria that in comparison to WT contained less than 5% and no detectable Cox activity, respectively.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0983
    Keywords: Yeast mutants ; Nitrogen mustard ; Thermoconditional DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Selection of mutants of Saccharomyces cerevisiae sensitive to the DNA cross-linking agent nitrogen mustard (HN2) at two temperatures (23 °C and 36 °C) yielded two isolates with thermoconditionally enhanced (ts) sensitivity to the mutagen. Both were due to single recessive nuclear genes. Mutant allele snm1–2 ts showed mainly ts-sensitivity to HN2, whereas mutant allele snm2-1 ts conferred ts-sensitivity to HN2, half mustard (HN1) and UV. In temperature-shift experiments it was determined that the functions of SNM1 and SNM2 are needed for recovery within 6 to 7 h. after mutagen exposure during incubation at 23 °C on YEPD when HN2 and UV are applied. After HN1 treatment the SNM2 coded function is required for recovery for about 14 hrs. This possibly indicates a handling of UV- and HN2-induced lesions different from that of HN1-induced lesions.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 231 (1992), S. 194-200 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; DNA repair ; Nitrogen mustard ; Interstrand cross-links ; Nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 3.2 kb yeast DNA fragment containing the DNA interstrand cross-link-specific repair gene SNM1 has been sequenced. Two genes were identified. SNM1 has an open reading frame of 1983 by and codes for a 661 amino acid protein. Hydrophobic analysis shows that the protein is most probably not directly membrane bound. The second gene, UGX1, has an open reading frame of 573 by coding for a polypeptide of 191 amino acid residues. The two genes are arranged head to head and share a 192 by divergent promoter region that contains three TATAAA motives, two for the SNM1 and one for the UGX1 locus. Gene UGX1 has no apparent influence on the sensitivity of the cell to cross-linking nitrogen mustard, as its disruption in wild type does not increase sensitivity to nitrogen mustard and the presence of multiple copies of the gene fails to complement the nitrogen mustard sensitivity phenotype of snm1 disruption mutants. Northern analysis revealed that the expression of SNM1 yields an average of 0.3 copies/cell of a 2.4 kb transcript, while expression of UGX1 yields higher levels of a 0.8 kb poly(A)+ RNA.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 241 (1993), S. 680-684 
    ISSN: 1617-4623
    Keywords: Nitrogen mustard resistance ; Regulation of choline permease ; Co-regulation ; Phospholipid biosynthesis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5′-CATGTGAAAT-3′) was found to be mandatory for CTR/HNM1 expression. This ‘decamer’ motif is located between nucleotides −262 and −271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of β-galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions −213 or −152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Formaldehyde hyper-resistance ; Alcohol dehydrogenase ; Glutathione ; Inducibility
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 3.7 kb DNA fragment of yeast chromosome IV has been sequenced that contains the SFA gene which, when present on a multi-copy plasmid in Saccharomyces cerevisiae, confers hyper-resistance to formaldehyde. The open reading frame of SFA is 1158 by in size and encodes a polypeptide of 386 amino acids. The predicted protein shows strong homologies to several mammalian alcohol dehydrogenases and contains a sequence characteristic of binding sites for NAD. Overexpression of the SFA gene leads to enhanced consumption of formaldehyde, which is most probably the reason for the observed hyper-resistance phenotype. In sfa:LEU2 disruption mutants, sensitivity to formaldehyde is correlated with reduced degradation of the chemical. The SFA gene shares an 868 by divergent promoter with UGX2 a gene of yet unknown function. Promoter deletion studies with a SFA promoter-lacZ gene fusion construct revealed negative interference on expression of SFA by upstream sequences. The upstream region between positons − 145 and − 172 is totally or partially responsible for control of inducibility of SFA by chemicals such as formaldehyde (FA), ethanol and methyl methanesulphonate. The 41 kDa SFA-encoded protein was purified from a hyper-resistant transformant; it oxidizes long-chain alcohols and, in the presence of glutathione, is able to oxidize FA. SFA is predicted to code for a long-chain alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) of the yeast S. cerevisiae.
    Type of Medium: Electronic Resource
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