Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.     

Energy-dependent mitochondrial mutagenicity of antibacterial ofloxacin and its recombinogenic activity in yeast

Ofloxacin, a specific inhibitor of bacterial topoisomerase II, is known to inhibit the growth of yeast cells and to induce rho ^– mutants in the yeast S. cerevisiae. The frequency of ofloxacin-induced petite mutants under non-growth conditions was found to be strongly diminished when the cells were depleted in intramitochondrial ATP. Under optimal conditions of mitochondrial mutagenesis the drug induced mitotic recombination and reverse mutation in diploid strains but failed to cure either killer plasmids or the 2 m DNA of dividing cells. The sensitivity to ofloxacin of the strains deficient in the DNA strandbreak repair pathway (rad52) was significantly higher then that of the wild-type strains and of the mutants deficient in excision or mutagenic DNA repair. The results are compatible with the idea that the cytotoxic and genetic activity of ofloxacin in yeast probably results from the inhibited DNA ligation function of topoisomerase II creating DNA breaks that are reparable through the recombination repair pathway.     

Heterologous gene expression of the glyphosate resistance marker and its application in yeast transformation

Summary The E. coli aroA gene was inserted between yeast promoter and terminator sequences in different shuttle expression plasmids and found to confer enhanced EPSP synthase activity as well as resistance to glyphosate toxicity. Subsequently, a transformation system using these newly constructed vectors in yeast was characterized. The efficiency of the glyphosate resistance marker for transformation and selection with plasmid pHR6/20-1 in S. cerevisiae laboratory strain SHY2 was found to be relatively high when compared with selection for LEU2 prototrophy. The fate of the recombinant plasmid pHR6/20-1 in the transformants, the preservation of the aroA E. coli DNA fragment in yeast, mitotic stability, EPSP synthase activity, and growth on glyphosate-containing medium have been investigated. As this plasmid also allows direct selection for glyphosate resistant transformants on rich media, the glyphosate resistance marker was used for transforming both S. cerevisiae laboratory strain SHY2 and brewer's yeast strains S. cerevisiae var. uvarum BHS5 and BHS2. In all cases, the vector pHR6/20-1 was maintained as an autonomously replicating plasmid. The resistance marker is, therefore, suitable for transforming genetically unlabeled S. cerevisiae laboratory, wild, and industrial yeast strains.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate     

Effect of ethidium bromide on transmission of mitochondrial genomes and DNA synthesis in the petite negative yeast Schizosaccharomyces pomhe

Summary Treatment of haploid strains of the petite negative yeast Schizosaccharomyces pomhe with ethidium bromide prior to mating with untreated cells reduces transmission of mitochondrial markers from the treated strains. This effect is fully reversible after 20 generations of growth in drug free medium before mating. In contrast to the petite positive yeast Saccharomyces cerevisiae, where nuclear DNA synthesis is not affected but mitochondrial DNA is degraded in the presence of 20 g/ml ethidium bromide, the same concentration decreases both nuclear and mitochondrial DNA synthesis in Schizosaccharomyces pomhe. After removal of the drug, nuclear DNA synthesis increases faster than its mitochondrial counterpart in Schizosaccharomyces pomhe.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

Polymorphism within the nuclear and 2μm genomes of Saccharomyces cerevisiae

Summary Seven strains of bakers' yeast were obtained as a representative sample of the Spanish baking industry. The nuclear genome was monitored for polymorphism by transverse alternating field electrophoresis (TAFE) and restriction maps of 2 m DNA were produced. All seven strains were uniquely different when evaluated by their total chromosomal lengths whereas only two 2 m variants were defined. There was no apparent correlation between chromosomal and plasmid polymorphism. The extensive chromosomal polymorphism within one 2 m DNA type indicates the rapid and relatively recent evolution of the nuclear genome. The hybrid origin (S. cerevisiae-S.monacensis) of lager yeast was critically evaluated by TAFE analysis of S. cerevisiae and S. carlsbergensis chromosomes. The absence of corresponding S. cerevisiae chromosomes III and XIII in S. carlsbergensis argued against the hybrid origin of lager strains. We discuss limitations of the hybrid origin hypothesis of industrial yeasts and propose that the molecular coevolution observed in 2 m DNA serves as a useful additional mechanism for rationalization of some of the structural polymorphism of the nuclear genome.     

Molecular properties of the lys1 + gene and the regulation of α-aminoadipate reductase in Schizosaccharomyces pombe

The -aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1 ⁺ gene and the regulation of the encoded -aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-EcoRI fragment of S. pombe DNA, containing a functional lys1 ⁺ gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented minimal or YEPD media was less than the activity of cells grown in minimal mediu. The AAR of S. pombe was more sensitive to feedback inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the -aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.     

New mutations in the yeast Saccharomyces cerevisiae affecting completion of “start”

Summary Here we report the isolation of several new temperature-sensitive mutations which cause cells of the yeast Saccharomyces cerevisiae to arrest in the G1 period of the cell cycle. Four different selection schemes were employed. The cell division cycle (cdc) mutations define five new complementation groups. At non-permissive temperatures, strains bearing these new cdc mutations arrested in G1 within one cell division cycle. By order-of-function mapping, cells of each population were found to be arrested at start, the regulatory point in the G1 period of yeast. Mutations were grouped into two categories by the abilities of mutant strains to continue extensive macromolecular synthesis and to conjugate with cells of the opposite mating type. For strains with mutations in one category, shift to the non-permissive temperature caused an abrupt decrease in the rates of labelling of protein and RNA, and rendered cells unable to mate efficiently. For strains with mutations in the second category, cells continued to grow and mating ability was not significantly impaired.Each selection scheme was also designed to isolate mutations which specifically affect the ability of cells to reinitiate the cell cycle from stationary phase. This was done to test the hypothesis that stationary phase cells are in a unique developmental state referred to as G0. No mutations specific for resumption of growth from stationary phase were isolated.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

pH sensitivity of Schizosaccharomyces pombe: effect on the cellular phenotype associated with lacZ gene expression

We report on a series of experiments inSchizosaccharomyces pombe to detect the blue-colour colony phenotype associated with expression of theEscherichia coli lacZ gene. Increasing the pH in solid minimal medium to optimize blue colony colour revealed a pH-sensitive phenotype in auxotrophic strains requiring uracil and leucine as external supplements. This phenotype was observed among commonS. pombe stock strains, 5-fluoroorotic acid (5-FOA)-selected strains, and random genetic segregants. Growth of prototrophicS. pombe strains 972 and 975 or the adenine auxotrophic strain NCYC 1860 were unaffected by an increase in external pH. Analysis of genetic segregants from three independent crosses indicated that a single auxotrophic marker (ura4 ^- orleu1-32) was sufficient for yeast cell-growth inhibition when the medium pH was increased above 6.6. In contrast, growth of aSaccharomyces cerevisiae strain isogenic to AH22, requiring uracil, leucine and histidine, was unaffected by changes in the pH of the medium. These observations suggest that uptake of uracil and leucine intoS. pombe cells is compromised by alterations in external pH. Our results have implications for detection of thelacZ gene-encoded bluecolour colony phenotype inS. pombe, which is optimized by growth in the presence of 5-bromo-4-chloro-3-indolyl--D-galactoside (Xgal) at pH 7.0. We discuss the conditions under which this blue-colour phenotype can be routinely observed inS. pombe.     

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Summary Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G₂ phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 m³). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 m³) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.Abbreviation AIR aminoimidazole ribonucleotide - DAPI 4,6-diamidino-2-phenylindole - PCA perchloric acid - DABA 3,5-diaminobenzoic acid     

Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

Summary The E. coli polA ⁺ gene has been subcloned from a specialised transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA⁺ gene was expressed. S. cerevisiae ars-1 or 2 replicative sequences were introduced into the polA⁺ plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2–20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA⁺ gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA⁺ gene.     

Starvation for His-tRNAHis in yeast causes translational arrest without a high level of misincorporation of glutamine at histidine codons

Summary The hts1.1 temperature-sensitive histidinyl-tRNA synthetase mutation enables Saccharomyces cerevisiae to be starved for His-tRNA^His by upshift to the non-permissive temperature of 38°C. If yeast behaves similarly to bacterial and mammalian cells, this lack of His-tRNA^His should greatly enhance misreading at histidine codons (CAU/CAC) by Gln-tRNA^Gln, resulting in substitution of the neutral amino acid glutamine in place of histidine, a basic amino acid. Such misreading causes the isoelectric point (pI) of proteins to shift to lower values, and is readily detectable as stuttering on two-dimensional (2D) protein gels. By gel analysis of pulse-labelled proteins of hts1.1 yeast cells that were overexpressing phosphoglycerate kinase (PGK), our study sought to detect this specific translational error in PGK protein. It was not detected by this relatively sensitive technique, indicating that missense errors due to glutamine insertion at histidine codons do not occur in yeast at the readily-detectable level found in bacterial and mammalian cells.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

Summary A general procedure for the curing of 2-m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -m DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-m DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-m DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir ⁺ and cir ⁰ strains has been compared.     

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.     

Organization of ribosomal RNA genes in Alternaria alternate Japanese pear pathotype,a host-selective AK-toxin-producing fungus

Summary DNA encoding ribosomal RNA (rRNA) of Alternaria alternata Japanese pear pathotype has been cloned in , replacement vector, , Fix. Restriction endonuclease mapping and Southern hybridization with the 18S and 28S rRNAs of Saccharomyces cerevisiae revealed the A. alternata rDNA to be tandemly repeating 8.15-kilobase pair unit. The restriction fragments of the unit were then subcloned in the plasmid vector Bluescribe M13- and partially sequenced. The determined sequences were compared with previously reported sequences of S. cerevisiae rRNAs and their genes. The locations of DNA sequences encoding the 5.8S, 18S, and 28S rRNAs were determined by homology search using reported sequences. The complete DNA sequence for 5.8S rRNA of the fungus was found to be highly conserved at more than 90 % homology in the fungi analyzed. However, sequence diversities were observed in limited regions involved in a helix structure, the helix (e), found at position 116–137.Deceased     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.