The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.     

The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase

Summary DNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD ⁺ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD ⁺ strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m⁵C residues.     

UV-induced curing of the double-stranded RNA virus of the yeast Yarrowia lipolytica

Summary Curing of the viruslike particles harbored by a strain of the yeast Yarrowia lipolytica was achieved by UV irradiation. The cured strain was found to be able to maintain the viruslike particles after their re-introduction by crossing or by cytoplasmic fusion. The involvement of a UV-induced mutation of a yeast maintenance gene seems therefore unlikely.Abbreviations UV ultraviolet - dsRNA double-stranded RNA - VLPs viruslike particles, A and B alleles of the mating type locus - TCA trichloracetic acid     

Base analogue mutagenesis in yeast and its modulation by pyrimidine deoxynucleotide pool imbalances: incorporation of bromodeoxyuridylate and iododeoxyuridylate

Summary Cells of the yeast, Saccharomyces cerevisiae, which are auxotrophic for thymidylate (tmp1) can also incorporate analogues of thymidylate. When the base analogue, 5-bromodeoxyuridylate, is incorporated into tmp1 yeast cells it is lethal and mutagenic. Both lethality and mutation induction can be drastically altered by perturbation of the pyrimidine nucleotide pools. Analysis of mutation induction, bromodeoxyuridylate incorporation into DNA, and cell viability under various conditions revealed: (1) lethality and mutagenesis can be uncoupled, (2) thymidylate enhances mutagenesis and deoxycytidylate suppresses it, (3) mutation induction is not correlated with the magnitude of bromodeoxyuridylate incorporation into DNA. Therefore, in yeast, the pyrimidine nucleotide pools have a powerful effect on bromodeoxyuridylate mutagenesis.Both bromodeoxyuridylate and iododeoxyuridylate are extensively incorporated into the DNA of tmp1 yeast cells; however, iododeoxyuridylate is non-mutagenic. Replication proceeds at the same rate in the presence of the natural substrate or either analogue. When cells are supplied with thymidylate and bromodeoxyuridylate together, there is no discrimination against bromodeoxyuridylate as a DNA precursor. However, in the presence of thymidylate and iododeoxyuridylate, there is a 3 to 1 discrimination against iododeoxyuridylate as compared to thymidylate.Abbreviations BU 5-bromouracil - BrdUrd bromodeoxyuridine - BrdUMP bromodeoxyuridylate - BrdUTP bromodeoxyuridine triphosphate - CDP cytidine diphosphate - dCyt deoxycytidine - dCMP deoxycytidylate - dCDP deoxycytidine diphosphate - dCTP deoxycytidine triphosphate - IdUrd iododeoxyuridine - IdUMP iododeoxyuridylate - dThd deoxythymidine - dTMP deoxythmidylate - dTTP deoxythymidine triphosphate     

Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes

HO-endonuclease initiates a mating-type switch in the yeast S. cerevisiae by making a doublestrand cleavage in the DNA of the mating-type gene, MAT. Heterothallic strains of yeast have a stable mating type and contain a recessive ho allele. Here we report the sequence of the ho allele; ho has four point mutations all of which encode for substitute amino acids. The fourth mutation is a leucine to histidine substitution within a presumptive zinc finger. Chimeric HO/ho genes were constructed in vivo by converting different parts of the sequence of the genomic ho allele to the HO sequence by gene conversion. HO activity was assessed by three bioassays: a mating-type switch, extinction of expression of an a-specific reporter gene, and the appearance of Can^r Ade^- papillae resulting from excision of an engineered Ty element containing the HO-endonuclease target site and a SUP4 ^o gene. We found that the replacement of the fourth point mutation in ho to the HO sequence restored HO activity to the chimeric endonuclease.     

Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase

Summary The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48 500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.     

Inhibition of thymidylate biosynthesis induces mitotic unequal sister chromatid recombination in Saccharomyces cerevisiae

Summary Inhibition of thymidylate biosynthesis has been found to induce deletion of a LEU2 insert from the ribosomal DNA gene cluster of haploid strains of Saccharomyces cerevisiae. Loss of the insert was detected phenotypically by the enhanced production of both sectored (leu⁺/leu^–) and non-sectored (leu^–) colonies. Hybridization patterns obtained by Southern blot analysis of DNA from the leu⁺ and leu^– sectors were consistent with the occurrence of unequal sister chromatid recombination. The induction of sectored colonies was prevented by the rad52-1 mutation but not by defects in RAD6. However, the formation of non-sectored leu^– colonies was induced by thymidylate depletion in both rad52-1 and rad6 strains.     

Genetic effects of photoactivated psoralens during meiosis in DNA repair mutantpso3-1 ofSaccharomyces cerevisiae

The influence of the DNA repair genePSO3 on photoactivated psoralen-induced meiotic recombination, gene conversion, reverse mutation, and on survival, was assayed in diploid strains ofSaccharomyces cerevisiae homozygous for the wild-type or thepso3-1 mutant allele. Sporulation was normal in thepso3-1 diploid. Wild-type and mutant strains had the same sensitivity to photoactivated monofunctional psoralen (3-CPs+UVA) in meiosis-uncommitted and meiosis-committed stages. The mutant showed higher sensitivity to photoactivated bifunctional psoralen (8-MOP+UVA) during all stages of the meiotic cycle. Mutation induction by 3-CPs+UVA or 8-MOP+UVA in meiosis-committed cells revealed no significant differences between wild-type and thepso3-1 mutant. The status of thePSO3 gene has no influence on the kinetics of induction of gene conversion and crossing-over after 3-CPs+UVA treatment in meiosis-committed cells: gene conversion was blocked while recombination was induced. After treatment with 8-MOP+UVA gene conversion was also blocked in both strains while crossing-over could only be observed in meiosis-committed wild-type cells.     

Mutations in ADE3 reduce the efficiency of the omnipotent suppressor sup45-2

Summary Mutations in a known yeast gene, ADE3, were shown to act as an antisuppressor, reducing the efficiency of the omnipotent suppressor, sup45-2. The ADE3 locus encodes the trifunctional enzyme C₁-tetrahydrofolate synthase, which is required for the biosynthesis of purines, thymidylate, methionine, histidine, pantothenic acid and formylmethionyl-tRNA^fmet. The role of this enzyme in translational fidelity had not previously been suspected.     

Genetic basis of the <Emphasis Type="Italic">ovc</Emphasis> phenotype of <Emphasis Type="Italic">Neurospora:</Emphasis> identification and analysis of a 77 kb deletion

The ovc mutant of Neurospora crassa accumulates more carotenoids than the wild type in the light, is sensitive to high osmotic pressure and exhibits an altered aerial development. The three traits are complemented by a single gene, cut-1, but only the two latter are exhibited by a mutant of this gene carrying a premature stop mutation. Targeted cut-1 deletion results in a normal carotenoid content, confirming the involvement of at least a second gene in the carotenoid-overproducing phenotype of the ovc strain. Molecular analysis of ovc genomic DNA indicates the absence of a large DNA segment affecting the gene cut-1. A PCR walking approach allowed the identification of a deletion extending along 77,078 bp on linkage group IV. The break-points are located in ApA/TpT sequences, suggesting the involvement of UV-induced thymine dimers in the origin of the deletion. The ovc mutant lacks 21 predicted ORFs, including cut-1 as the only known genetic marker, and four ORFs from a 22-member transmethylase gene family. Ten ORFs have no similarity with any predicted gene from other species. Three of them are closely related by sequence and linkage, evoking ancestral gene duplications.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

Characterization of the <Emphasis Type="Italic">Arxula adeninivorans AHOG1</Emphasis> gene and the encoded mitogen-activated protein kinase

Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol. As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway. In the present study, we isolated and characterized as a key component of this pathway the A. adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa. The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron. The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi. Under stress-free conditions, the inactive Ahog1p is present in low levels. When exposed to osmotic stress, Ahog1p is rendered active by phosphorylation. In addition, AHOG1 expression is increased. Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species. This specific property could account for the fast adaptation and high osmo-resistance encountered in this species.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Failure to detect an antimutator phenotype following disruption of the Saccharomyces cerevisiae DDR48 gene

The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway. We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator. However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2–1 allele, or forward mutation at the CAN1 locus. Interestingly, the latter gene had been reported previously to be subject to the antimutator effect. DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes. Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype.     

Isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae by functional complementation in yeast

Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment.By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

The C-terminal part of the CDC25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-CDC25 protein

The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity. A chimera between the 3 regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal CDC25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the CDC25 and the SDC25 gene products are structurally and functionally similar. The CDC25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.     

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase

Summary The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.