GSG1, a yeast gene required for sporulation

We have identified a gene, GSG1 (g eneral s porulation g ene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and α haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4–6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication. The sequence reported here has the GenBank Accession Number U26674.     

Genomic disruption of six budding yeast genes gives one drastic example of phenotype strain-dependence

Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.     

Isolation of Meiotic Segregants from a Bottom Fermenting Yeast

Viable meiotic segregants were isolated from a bottom fermenting brewing yeast transformed by IME1 using a high copy number plasmid. These transformants sporulated under sporulation conditions and viable meiotic segregants were obtained from the spore asci. Flow cytometry showed that the meiotic segregants had a lower DNA content per cell than the parental strains, indicating meiosis had proceeded normally. Chromosomal observation by pulsed‐field gel electrophoresis and array comparative genomic hybridization (array‐CGH) showed that meiotic segregants, like the parent strains, contained two types of chromosome: the S. cerevisiae type and the S. bayanus type. In certain meiotic segregants, some chromosomes were missing, and the chromosome copy number changed. Isolation of meiotic segregants resulted in a ploidy reduction, which can be applied in the breeding of yeast strains.     

Control of division arrest and entry into meiosis by extracellular alkalisation in Saccharomyces cerevisiae

Limitation of nutrients allows yeast cells to arrest proliferation at G1 phase of the cell cycle and to enter the so-called stationary phase. We show here another pathway for cytostasis, which is associated with extracellular accumulation of bicarbonate and the resulting alkalisation of medium during the proliferation of cells respiring acetate. Alkalisation of medium by addition of bicarbonate or alkaline buffers ceased proliferation at G1 phase of logarithmically growing cells and caused a severe drop in G1-cyclin (CLN1 and CLN2) mRNAs. The arrested cells were heat-shock resistant, suggesting that the cells entered the stationary phase. Cells confluently grown on acetate re-entered into the cell cycle after acidification of the culture medium. These results indicate that external alkalisation is a primary cause of the cytostasis. The alkali-induced G1 arrest was shown to be cyclic AMP (cAMP)-independent using mutant cells which lack a functional Ras/cAMP signaling pathway. Alkalisation of medium also stimulated meiosis and sporulation in rich acetate medium, confirming our previous proposal that environmental alkalisation but not nitrogen limitation is a key condition for entry into meiosis and sporulation. © 1998 John Wiley & Sons, Ltd.     

Functional interrelationships between carbohydrate and lipid storage,and mitochondrial activity during sporulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae under conditions of nutrient stress, meiosis precedes the formation of spores. Although the molecular mechanisms that regulate meiosis, such as meiotic recombination and nuclear divisions, have been extensively studied, the metabolic factors that determine the efficiency of sporulation are less understood. Here, we have directly assessed the relationship between metabolic stores and sporulation in S. cerevisiae by genetically disrupting the synthetic pathways for the carbohydrate stores, glycogen (gsy1/2Δ cells), trehalose (tps1Δ cells), or both (gsy1/2Δ and tps1Δ cells). We show that storage carbohydrate-deficient strains are highly inefficient in sporulation. Although glycogen and trehalose stores can partially compensate for each other, they have differential effects on sporulation rate and spore number. Interestingly, deletion of the G₁ cyclin, CLN3, which resulted in an increase in cell size, mitochondria and lipid stores, partially rescued meiosis progression and spore ascus formation but not spore number in storage carbohydrate-deficient strains. Sporulation efficiency in the carbohydrate-deficient strain exhibited a greater dependency on mitochondrial activity and lipid stores than wild-type yeast. Taken together, our results provide new insights into the complex crosstalk between metabolic factors that support gametogenesis.     

A Candida albicans gene encoding a DNA ligase

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession Number X95001.     

Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: Ultrastructural analysis of spo mutants

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.     

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium–glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2⁺ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat-shocked cells (38°C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.     

Sequencing and functional analysis of a 32 560 bp segment on the left arm of yeast chromosome II. Identification of 26 open reading frames,including the KIP1 and SEC17 genes

We report here the DNA sequence of a segment (α1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32·5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc-finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.     

FEN2: A gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast

We have isolated and characterized a pleiotropic recessive mutation, fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5·5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.     

Characterization of the SAC3 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC3 gene. A DNA fragment containing the SAC3 gene was sequenced. SAC3 codes for a 150 kDa hydrophillic protein which does not show any significant similarities with other proteins in the databases. Sac3 therefore is a novel yeast protein. A nuclear localization of Sac3 is suggested by the presence of a putative nuclear localization signal in the Sac3 sequence. A SAC3 disruption mutation was constructed. SAC3 disruption mutants were viable but grew more slowly and were larger than wild-type cells. In contrast to the sac3-1 mutation, the SAC3 disruption was not able to suppress the temperature sensitivity and the osmosensitivity of the act1-1 mutant. This demonstrates that act1-1 suppression by sac3-1 is not the result of a simple loss of SAC3 function. Furthermore, we examined the act1-1 and the sac3 mutants for defects in polarized cell growth by FITC-Concanavalin A (Con A)-labelling. The sac3 mutants showed a normal ConA-labelling pattern. In the act1-1 mutant, however, upon shift to non-permissive temperature, newly synthesized cell wall material, instead of being directed towards the bud, was deposited at discrete spots in the mother cell.     

CaRch1p does not functionally interact with the high‐affinity Ca2+ influx system (HACS) of Candida albicans

The plasma membrane protein CaRch1p of Candida albicans, homologous to the human solute carrier protein SLC10A7, is involved in the regulation of calcium homeostasis. C. albicans cells lacking CaRCH1 are hypersensitive to high extracellular Ca²⁺ concentrations and show increased tolerance to ketoconazole (KCZ). We assume a higher basal Ca²⁺ influx in the rch1/rch1 mutant strain at low extracellular Ca²⁺ concentrations, which is not detrimental to C. albicans cells but may be sufficient to activate calcineurin, finally resulting in an increased tolerance to KCZ. However, at 8 µg/ml KCZ plus 3 mm Ca²⁺ the rch1/rch1 mutant and the wild‐type strains showed identical growth. By further increasing the Ca²⁺ concentration to 30 mm , this phenotype was completely reversed and the rch1/rch1 mutant strain became extremely sensitive to 8 µg/ml KCZ, probably due to synergistic toxic effects of Ca²⁺ and KCZ under these conditions. Furthermore, we aimed to clarify whether CaRch1p interacts with the Cch1p component of the voltage‐gated calcium influx channel Cch1p/Mid1p in C. albicans cells. As disruption of the two alleles of CCH1 in the rch1/rch1 mutant strain did not alter its hypersensitivity to high extracellular Ca²⁺, and as this phenotype was completely abolished by low amounts of Mg²⁺ in the rch1/rch1 mutant as well as in the cch1/cch1 rch1/rch1 double mutant, we conclude that CaRch1p is a functional component of the low‐affinity calcium uptake system (LACS) system and does not functionally interact with Cch1p. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of the gene encoding scHelI,a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a |glgt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable of mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability. © 1997 John Wiley & Sons, Ltd.