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We previously identified four nuclear genes ( caf1+– caf4+) in Schizosaccharomyces pombe, mutations in which confer resistance to caffeine and brefeldin A. caf1+, caf2+ and caf4+ were sequenced and found to be identical to the multidrug-resistance/stress-response genes hba1, crm1 and trr1, respectively. Here we show that caf3 is allelic to pap1, which encodes an AP-1-like transcription factor. The allele associated with caffeine resistance, caf3-89 , contains a single-nucleotide exchange that results in a Leu?Ser exchange in the NES (nuclear export signal) domain of the gene product. Due to this alteration, the modified protein can not be exported from the nucleus back into the cytoplasm, and thus accumulates in the nucleus. The activity of pap1/caf3 is shown to be necessary for manifestation of the caffeine resistance caused by mutations in the genes hba1/caf1 and crm1/caf2. We also cloned two genes that confer caffeine resistance when carried on a multicopy plasmid. One of them turned out to be a truncated allele of pad1/bfr2/sks1, which codes for a subunit of the 26 S proteosome. The putative product of the other gene, designated caf5, has a structure highly similar to that of MFS permeases. It contains two groups of six transmembrane spanning domains each, with the conserved motifs WRW, PET and GAIGGPVLGP in the fifth and sixth domains. These results are all consistent with our earlier hypothesis, which suggested that the caf genes are functionally interlinked in a complex detoxification mechanism. caf5 and pad1 may also encode parts of this mechanism. more...

Abstract. We have previously described the genetic analysis of eleven complementation groups (sep6-sep16) defined by Schizosaccharomyces pombe mutants that are defective in cell separation and sexual differentiation. Here we report on the cloning and characterisation of two members of this set, sep10 and sep11. Sequencing of the full-length sep10 revealed a continuous ORF that encodes a conserved protein with possible functions in general transcriptional regulation. The coding region of sep11 is interrupted by introns and the putative sep11 protein shows no sequence similarity with known proteins of other species. Disruption of each gene causes temperature sensitivity. Simultaneous disruption of both genes is lethal, demonstrating that sep10 and sep11 perform related, overlapping functions. Overexpression of aff1/ste11, a pivotal regulator of sexual development, suppresses the sterility of sep10^- cells, which suggests that sep10 is needed for the activity of aff1/ste11. more...

The dimorphic fission yeast Schizosaccharomyces japonicushas proved to be an excellent experimental model for the investigation of the eukaryotic cell. Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote. To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as arswhen integrated into a plasmid. Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion. The complementation tests and the recombination frequencies determined in octads of spores identified 28 non‐allelic groups (genes) of mutations of the auxotrophic and mycelium‐negative mutants. Two groups of linked markers were also identified. The cloned fragment, which expresses arsactivity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae). Copyright © 2002 John Wiley & Sons, Ltd. more...

The proper division of cells is essential for the production of viable daughter cells. In plants and fungi, the dividing cell produces a cross-wall or septum that bisects the cytoplasm. For separation of the daughter cells, the septum has to be cleaved. To study the regulation of this process, we isolated mutants defective in septum cleavage. The mutants showed highly pleiotropic phenotypes and defined 17 novel genes. The deduced amino acid sequences of the products of the cloned genes exhibited homologies to various transcription regulators of other organisms. The homologies and the pleiotropic effects of the mutations on sexual development, stress response, mitotic stability, septum initiation and septum placement indicated that these genes affect cell separation indirectly, through multifunctional regulatory modules. more...

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomycesyeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomycessensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control. more...

Genetic analysis of 20 sterile mutants prone to form hyphae revealed 11 novel stegenes (sep6to sep16) of Schizosaccharomyces pombe. None of the mutants was completely mycelial. Most mutants formed branching hyphae and showed normal septation. Aberrant septal structures and actin distribution were seen only at 36°C. sep9‐307, sep14‐576and sep15‐598showed genetic interactions with sep1‐1, a mutation in a forkhead transcription factor homologue. Additional genetic interactions were detected between sep6‐194, sep15‐598and cdc16‐116, a mutant allele of an anaphase modulator of p34cdc2. sep9‐307and sep15‐598caused dikaryosis in wee1−background. In mating and sporulation tests, sep6−, sep7−, sep9−, sep10−, sep11−and sep15−proved to be defective in conjugation only, whereas sep8−, sep13−and sep16−were also defective in meiosis‐sporulation. sep12−and sep14−were only partially sterile. All mutants could produce M‐factor but sep8−, sep11−, sep15−and sep16−were defective in P‐factor production. The mutations in sep8, sep11and sep16suppressed the pat1‐114‐driven meiosis. All mutants were sensitive to the presence of higher concentrations of chloride in the medium and to short heat shocks. The diversity of the mutant phenotypes and the pleiotropic effects of the mutations suggest that these sepgenes might act in, or interact with, a multiple overlapping network of regulatory modules. Copyright © 1999 John Wiley & Sons, Ltd. more...

Hybrids were constructed between auxotrophic mutants of a heterothallic distiller's strain and a homothallic wine yeast. The hybridization resulted in a significant increase in both ethanol production and tolerance against exogenous ethanol. The hybrids were heterogeneous in ploidy, probably due to segregation of aneuploids during culturing. Sporulation of the hybrids broke down the high productivity, producing spore clones that were mostly of various intermediate levels of performance. However, a meiotic product superior to both crossing partners was also found. The results demonstrate that fermentation capacity can be improved by crossing with a low performance strain. more...

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl2, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed. more...

Mutation in the gene sep1+ of the unicellular fission yeast Schizosaccharomyces pombe impairs cell separation after cytokinesis and confers a branching mycelial morphology. The mutant is not defective in cell wall beta-glucanase activity but shows increased sensitivity to Ca2+ and Mg2+, and increased resistance to the microtubule inhibitor benomyl. The mycelial growth of sep1-1 provides a convenient method for the examination of the polar growth pattern and for pedigree analysis as demonstrated by the segregation of mating types in the homothallic microhyphae. sep1 is closely linked to ade1 (0.94 cM) on the right arm of chromosome II. The ts mutation spl1-1 confers a bent cell shape and causes aberrant septum formation at the restrictive temperature. sep1+ and spl1+ perform closely related functions as their mutant alleles interact with each other and with another septation mutant cdc4-8. These functions may overlap with certain cytoskeletal processes and with the determination of cell polarity because the triple mutant forms huge multinucleate syncytia with promiscuous branching and rare septum formation. more...

Haploid homothallic strains of Schizosaccharomyces pombe with mutations in any of nine “sterility genes” (ste) do not mate with wild-type fertile strains. Those defective in genes ste1 to ste4 and ste7 to ste9 are also deficient in meiosis and sporulation. I found that the ste1, ste3 and ste8 genes act very early in the sexual development, presumably before the pat1-controlled conjugation-specific event. ste5 and ste6 exert their function downstream of pat1 in the initiation of conjugation and do not play any role in the meiotic pathway. ste2, ste4, ste7 and ste9 are involved in both sexual pathways: they seem to act downstream of pat1 in conjugation but upstream of pat1 in the initiation of meiosis. A new gene, aff1, whose defective allele suppresses the pat1-114-provoked haploid sporulation and arrest of vegetative growth is also described. It is supposed that the aff1+ gene product participates in a cascade of regulatory events, as a factor antagonistic to pat1. more...

The phylogenesis of fungi is controversial due to their simple morphology and poor fossilization. Traditional classification supported by morphological studies and physiological traits placed the fission yeasts in one group with ascomycetous yeasts. The rRNA sequence comparisions, however, revealed an enormous evolutionary gap betweenSaccharomyces andSchizosaccharomyces. As shown in this review, the protein sequences also show a large gap which is almost as large as that separatingSchizosaccharomyces from higher animals. Since the two yeasts share features (both cytological and molecular) in common which are also characteristic of ascomycetous fungi, their separation must have taken place later than the sequence differences may suggest. Possible reasons for the paradox are discussed. The sequence data also suggest a slower evolutionary rate in theSchizosaccharomyces lineage than in theSaccharomyces branch. In the fission yeast lineage two ramifications can be supposed. FirstS. japonicus (Hasegawaea japonica) branched off, thenS. octosporus (Octosporomyces octosporus) separated fromS. pombe. more...

Mutation in the gene sep1+ of the unicellular fission yeast Schizosaccharomyces pombe impairs cell separation after cytokinesis and confers a branching mycelial morphology. The mutant is not defective in cell wall beta-glucanase activity but shows increased sensitivity to Ca2+ and Mg2+, and increased resistance to the microtubule inhibitor benomyl. The mycelial growth of sep1-1 provides a convenient method for the examination of the polar growth pattern and for pedigree analysis as demonstrated by the segregation of mating types in the homothallic microhyphae. sep1 is closely linked to ade1 (0.94 cM) on the right arm of chromosome II. The ts mutation spl1-1 confers a bent cell shape and causes aberrant septum formation at the restrictive temperature. sep1+ and spl1+ perform closely related functions as their mutant alleles interact with each other and with another septation mutant cdc4-8. These functions may overlap with certain cytoskeletal processes and with the determination of cell polarity because the triple mutant forms huge multinucleate syncytia with promiscuous branching and rare septum formation. more...

Abstract: Protoplasts of auxotrophic mutants isolated fromSchizosaccharomyces pombe andSchizosaccharomyces octosporus were fused. The fusion products were capable of complementation and growth on minimal medium, but the morphological observations suggested a disturbed physiological balance, which was reflected in low viability, osmotic sensitivity, and the formation of incomplete cell wall. Their asymmetric segregation indicated thatSchizosaccharomyces octosporus might be dominant in the fused cells. more...

We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis. more...

Protoplasts of methionine-and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus. more...

The ligase-defective cdc17-L16 mutant of Schizosaccharomyces pombe var. pombe was tested for genetic recombination and mating-type switching. Mitotic recombination was studied in both haploid and heteroallelic diploid cells. Cells carrying a heteroallelic ade6 duplication constructed by Schuchert and Kohli were tested for ectopic genetic recombination. We have found that cdc17-L16 is a mitotic hyper-rec mutant, as it increases the instability of the duplication by a factor of about 6 even at the permissive temperature of 23° C. In diploid cells, the enhancement of recombination rates detected was to that of cdc17+ cells. The temperature-sensitive cell cycle defect is also associated with a reduced level of mating and sporulation but does not significantly affect mating-type switching and intragenic meiotic recombination. It is supposed that the mitotic hyper-rec phenotype is a secondary result of insufficient repair of DNA breaks, while the lack of influence of the reduced ligase activity on the latter two processes might be attributed to their peculiar initiation mechanisms. more...

The fission yeast Schizosaccharomyces pombe has a haplontic life cycle in which the diplophase is confined to the zygote. Through the use of one- and two-step protoplast fusions we show that the ploidy can be increased up to pentaploid. The polyploid fusion products are rather unstable and segregate cells of lower ploidies by gradual loss of chromosomes during mitotic divisions. The polyploid cells conjugate normally but are prone to arrest at various stages of meiosis (1-, 2- and 3-spored asci, binucleate spores) and/or produce inviable, most probably aneuploid, spores. Marker segregation in the complete tetrads indicates the multiple association of homologous chromosomes. In tetra- and penta-ploid meiosis, multispored (6- to 7-spored) asci are also produced, probably by postmeiotic division of the nuclei. more...

Cell division cycle mutants defective in G1, DNA replication or nuclear division were tested for sporulation at semi-restrictive temperatures. In cdc1-7, cdc5-120, cdc17-L16 and cdc18-46 no abnormalities were observed; cdc10-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with a low efficiency; cdc22-M45 was completely defective in meiosis, but could conjugate and formed zygotes with a single nucleus. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc13 were blocked in meiosis II. None of the wee1-50, adh.nim1+ and win1+ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis. The meiotic function of cdc13 is TBZ-sensitive and probably exerted downstream of cdc2. Single mutants in cut1 or cut2 did not effect sporulation, whereas the double mutant cut1 cut2 formed two-spored asci. The results demonstrate that the cell division cycle and the meiotic developmental pathway share common genes and regulatory cascades. more...

We isolated two sterile mutants of Schizosaccharomyces pombe. One of them was mapped close to ste13 (8 cM). Since it turned out to be allelic with the hitherto unmapped ral2, its linkage with ste13 localizes ral2 on the right arm of chromosome II. The other mutant defines a novel class-I ste gene, ste15, closely linked to ste7 (4 cM) on chromosome I. ste15 is conjugation-specific and acts upstream of pat1 and ras1. During its genetic analysis, a phenotypic suppression of ste12-N9 was observed which was caused by mutations in the unlinked gene ssw1. more...

A stable mutant having increased resistance to growth inhibition by caffeine was obtained from Schizosaccharomyces pombe after UV-irradiation of vegetative cells. The caf1-21 mutation confers increased UV-sensitivity, impaired fertility and sporulation, lengthened cell cycle and a slightly abnormal cell morphology, but it does not affect ectopic recombination in vegetative cells. The mutation is partially dominant and defines a gene mapping on chromosome II. Various possible mechanisms of resistance are discussed. more...

Although some differences are remarkable, the sexual system of Schizosaccharomyces pombe var. malidevorans is, in principle, compatible with that of Schizosaccharomyces pombe var. pombe. It has two heterothallic types analogous to Leupold's h+ h-, and a homothallic type, called spotty, which resembles h90. In addition, pseudoheterothallic types (pseudo-h- and pseudo-h+) similar to certain mutants of S.p.v. pombe defective in conjugation were also found, as well as a homothallic type (grey) which has not been described in S. p. v. pombe. Genetic analysis of various hybrids constructed by mating and protoplast fusion revealed the probable genetic determination of the sexual types. Assuming a mating-type region similar to that of S.p.v. pombe, a five-gene-system can be supposed. The mat-locus is the residence of the expressible mating type information (either plus or minus), one gene determines whether the clone is homo- or heterothallic, another one regulates the intensity of mating and sporulation, and two are responsible for accomplishing certain mating-type dependent conjugation functions. more...

ABSTRACTMinimal inhibitory and minimal fungicidal concentrations of caspofungin were determined for 48 Candida inconspicuaisolates. By using CLSI (formerly NCCLS) methodology with the partial inhibition endpoint criterion, caspofungin exhibited a good fungicidal effect against C. inconspicua(the MIC90was 0.25 μg/ml and the minimum fungicidal concentration [MFC] was 0.5 μg/ml after 24 h). Total inhibition yielded falsely elevated MICs, exceeding even the respective MFCs. more...

The average transmission-and recombination frequencies of mitochondrial markers are similar in diploid clones derived from zygotes or from fusion of haploid protoplasts of identical mating type. The transmissional patterns of mitochondrial markers in individual fusion-or zygote-clones, however, are very different. The time needed for regeneration of cell walls of fused protoplasts is found to be mainly responsible for this difference, since delay of first cell division in zygotes leads to similar results. more...

The fission yeastcdc2 gene is pleiotropic, functioning both in the cell division cycle and in meiosis. Here we show thatcdc2 is allelic totws1, a previously isolated meiotic gene. Dissociation of meiotic and mitotic roles of the gene is also demonstrated by finding mutant alleles specifically altered in only one of the two processes. more...

The cell-cycle start gene, cdc2+, and its activator, cdc25+, are required for the initiation of the second meiotic nuclear division. They exert their functions downstream of mes1, a gene presumed to act between meiosis I and meiosis II. weel+, the inhibitor of cdc2+ in mitotic control, does not appear to play any role in this process. more...

SEVERAL procedures for enrichment of fungal mutants are known and have been reviewed1,2but they have many limitations. Here we describe a new technique based on selective cell-wall lysis, which has wide applicability and is highly efficient for isolating mutants differing from the wild-type strain in growth requirements. more...