α-mating-type-specific suppression and a-mating-type-specific enhancement by tunicamycin of sexual agglutinability in the yeast Saccharomyces cervisiae

Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of pheromone which induces or enhances the sexual agglutinability of a cells.Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.Abbreviations AI agglutination index - TM tunicamycin     

Three aldo–keto reductases of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote. The S. cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes. We have identified several open reading frames in the S. cerevisiae genome which show significant similarity to members of the aldo–keto reductase superfamily. The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways. Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined. Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined.     

Inactivation of exogenous β-lactamase in transformed yeast Saccharomyces cerevisiae

Summary The stability of bacterial -lactamase in transformedSacharomyces cerevisiae grown on glucose was studied. A culture of a prototrophic strain showed marked inactivation shortly before the stationary phase. This was also observed in cells starved of nitrogen. The level of reserve carbohydrate was lower both in the stationary-phase culture of the auxotroph and in the glucose-starved culture of the prototroph, where less inactivation was observed. Such a close correlation suggests that inactivation may be triggered mainly in response to nitrogen-limitation which regulates reserve carbohydrate metabolism.     

Effect of anticalmodulin drugs on the action of yeast α factor pheromone

Saccharomyces cerevisiae factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of factor on MAT a cells.Abbreviation MAT mating type     

Cosntruction of the physical map of yeast（Saccharomyces cerevisiae）chromosome V~1

There are about 17 chromosomes in yeast Saccharomycescerevisiae.A middle sized chromosome,chromosome V,waschosen in this work for studying and constructing the physi-cal maps.Chromosome V from strain A364a was isolatedby pulsed-field gradient gel electrophoresis(PFGE).Gelslices containing chromosome V DNA were digestedwith two rare cutting enzymes,NotⅠand SfiⅠ,and three6-Nt recognizing enzymes,SmaⅠ,SstⅡ and ApaⅠ.Several strategies-partial or complete digestions,digestion with different sets of two enzymes,and hybrid-ization with cloned genetically mapped probes(CAN1,URA3,CEN5,PRO3,CHO1,SUP19,RAD51,RAD3)——were used to align the restriction fragments.There are 9,9,15,17,and 20 sites for NotⅠ,SfiⅠ,SmaⅠ,SstⅡ and ApaⅠrespectively in the map of the A364a chromosome V.Itstotal length was calculated to be 620 Kb(Kilo-bases).Thedistributions of the cutting sites for these five enzymesthrough the whole chromosome are not uniform.A comp-arison between the physical map and the genetic map wasalso made.     

Is DNA damage the signal for induction of thermal resistance? induction by radiation in yeast

Yeast, as well as higher eukaryotes, are induced to increase thermal resistance (thermotolerance) by prior exposure to a heat stress. Prior exposure to an acute dose of either 60Co gamma or 254-nm ultraviolet radiation, at sublethal or fractionally lethal doses, is shown to cause a marked increase in the resistance of Saccharomyces cerevisiae to killing by heat. Following a radiation exposure, thermal resistance increased with time during incubation in nutrient medium, and the degree of resistance reached was proportional to the dose received. Partial induction by radiation followed by maximum induction by heat did not produce an additive response when compared to a maximum induction by heat alone, suggesting that the same process was induced by both heat and radiation. Irradiation with 254-nm uv light followed by an immediate, partial photoreversal of the pyrimidine dimers with long-wavelength uv light resulted in a reduced level of resistance compared to cells not exposed to the photoreversal light, indicating that the cells specifically recognized pyrimidine dimers as a signal to increase their thermal resistance. Exposure to 254-nm uv or ionizing radiation induced thermal resistance in mutants defective in either excision repair (rad3, uv-sensitive) or recombinational repair (rad52, gamma-sensitive), suggesting that recognition and repair of DNA damage by these systems are not a part of the signal which initiates an increase in resistance to heat. The amount of induction, per unit dose, was greater in the DNA repair-deficient mutants than in the wild-type cells, suggesting that an increase in the length of time during which damage remains in the DNA results in an increase in the effectiveness of the induction. These data indicate that types of DNA damage as diverse as those produced by ionizing radiation and by ultraviolet light are recognized as a signal by the yeast cell to increase its thermal resistance. It is therefore suggested that heat-induced alterations in DNA or in DNA-dependent chromosomal organization may be the signal for heat induction of thermotolerance in this and other eukaryotes.     

Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.     

Vacuoles: The sole compartments of digestive enzymes in yeast (Saccharomyces cerevisiae)?

Almost all the vacuoles (about 95%) remained intact after polybase-induced lysis of the yeast protoplasts. These vacuoles could be sedimentated together with other cell organelles which were equally well preserved, leaving as a supernatant a cytosol fraction which was essentially uncontaminated by the contents of disrupted vacuoles. After density gradient centrifugation more than half of the vacuoles were recovered in a fraction which was highly purified as judged from the measurement of several marker enzymes and from light and electron microscopic observations. Polyphosphate, which has been shown to be located exclusively in the vacuolar sap of protoplasts, was used as a vacuolar marker to determine the yields of vacuoles in the different fractions obtained from the density gradients. It was also used to assess the overall distribution of lytic enzymes in the cytosol and in the vacuome.The results indicate that the following enzyme activities are mostly, if not exclusively (>90%), located in the vacuome, probably all in the typical large vacuoles present in the protoplasts: exo-and endopolyphosphatase, proteases A and B, carboxypeptidase Y, an aminopeptidase, RNase, -mannosidase, and phosphatases which hydrolyze a number of different substrates. The polyphosphatases are thus in the same compartment as the polyphosphate. The activities of some other hydrolases, notably of a Mg²⁺ dependent, Oligomycin and NaN₃ insensitive ATPase and alkaline phosphatase, were partially associated with the vacuoles. The activities of pyrophosphatase, tripolyphosphatase, -glucosidase, and aminopeptidase active in the presence of EDTA, were located almost exclusively in the soluble, cytosolic fraction.Non-Standard Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - BSA bovine serum albumin - BTNA N-benzoyl-L-tyrosine-p-nitroanilide - LeuNA leucine-p-nitroanilide - LysNA lysine-p-nitroanilide - PIPES pinerazine-N,N-bis-2-ethanesulfonic acid - PP polyphosphate - Tri-PP tripolyphosphate     

Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae

A recombinant plasmid, pGMF, containing a gamma-glutamylcysteine synthetase gene (GSH-I) from Saccharomyces cerevisiae, was constructed with a copper-resistance gene as the selection marker and was introduced into S. cerevisiae YSF-31. The glutathione content of the recombinant strain was 1.5-fold (13.1 mg g dry cells(-1)) of that in the host strain.     

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.     

Isolation and amino acid sequence of a mating pheromone produced by mating type α cells of Saccharomyces exiguus

A peptide, termed α^se pheromone, was isolated as a mating pheromone from culture filtrate of mating type a cells of Saccharomyces exiguus. The peptide showed both agglutinability-inducing activity to a cells of S. cerevisiae and shmoo-inducing action to a cells of S. cerevisiae, S. kluyveri and S. exiguus. The amino acid sequence of α^se pheromone was determined as H-Trp-His-Trp-Leu-Arg-Leu-Ser-Tyr-Gly-Gln-Pro-Ile-Tyr-OH by mass spectrometry, sequence analysis and enzymatic digestion.     

Contribution by Saccharomyces cerevisiae yeast to fermentative flavour compounds in wines from cv. Albariño

A comparative study was made of the fermentation products of Spanish Albariño wines produced with spontaneous yeast flora and an indigenous selected Saccharomyces cerevisiae strain (Alb16). The content of fermentative volatile compounds was determined by gas-chromatography-FID. Fifteen compounds (5 alcohols, 7 esters and 3 acetates) were identified in the two Albariño wines studied. Higher alcohols, ethyl esters (except ethyl hexanoate and ethyl octanoate) and acetates were in greater concentration in the spontaneous fermentation wine than in that with selected Alb16 strain. Principal components analysis showed good separation between the different wines.     

Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation–dissociation

Spatial and temporal behavior of chromosomes and their regulatory proteins is a key control mechanism in genomic function. This is exemplified by the clustering of the 32 budding yeast telomeres that form foci in which silencing factors concentrate. To uncover the determinants of telomere distribution, we compare live-cell imaging with a stochastic model of telomere dynamics that we developed. We show that random encounters alone are inadequate to produce the clustering observed in vivo. In contrast, telomere dynamics observed in vivo in both haploid and diploid cells follows a process of dissociation–aggregation. We determine the time that two telomeres spend in the same cluster for the telomere distribution observed in cells expressing different levels of the silencing factor Sir3 protein, limiting for telomere clustering. We conclude that telomere clusters, their dynamics, and their nuclear distribution result from random motion, aggregation, and dissociation of telomeric regions, specifically determined by the amount of Sir3.     

Differences in glucose sensing and signaling for pexophagy between the baker’s yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris

The mechanism(s) of glucose sensing for inducing the autophagic peroxisome degradation (pexophagy) is not known. Recently, we have found that defects in the S. cerevisiae PKA-cAMP signaling pathway due to knockouts of GPR1 and/or GPA2 suppressed glucose-induced degradation of peroxisomal thiolase. Here we report that single defects of high (SNF3) and low (RGT2) affinity glucose sensors involved in glucose-dependent induction of hexose transporters have only a slight effect on glucose-induced degradation of peroxisomal thiolase, although simultaneous defects of both sensors, SNF3 and RGT2 (which are known to strongly affect glucose transport) strongly inhibit this process in S. cerevisiae. Most likely, glucose is sensed for pexophagy using the Gpr1 sensor involved in the PKA-cAMP signaling pathway. In the methylotrophic yeast P. pastoris, however, knock out of S. cerevisiae orthologs of GPR1 and GPA2 did not affect glucose-induced degradation of oleate-induced thiolase or the methanol-induced key peroxisomal protein, alcohol oxidase. This implies that glucose sensing for pexophagy is different in baker’s and methylotrophic yeasts.

Addendum to: Nazarko VY, Thevelein JM, Sibirny AA. G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae. Cell Biol Int 2007; doi:10.1016/j.cellbi.2007.11.001.     

Isolation of a Peptidyl Factor, α Substance-IA,Inducing Sexual Agglutinability in Saccharomyces cerevisiae

After a eleven-steps purification, a peptidyl factor named α substance-Ia was isolated in pure form from a culture filtrate of α type cells of the heterothallic Saccharomyces cerevisiae. The substance induced the sexual agglutinability in a haploid cells belonging to the opposite mating type at concentrations from 0.4 to 0.8 ng/ml.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.