New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines

The fermentation of grape must using non‐Saccharomyces yeasts with particular metabolic and biochemical properties is of growing interest. In the present work, red grape must was fermented using four strains of Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar (glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.     

Physiological and biochemical analysis of cytochrome P-450 in the yeast Schizosaccharomyces pombe

The yeast Schizosacchromyces pombe has been shown to contain a microsomal cytochrome P-450 (cyt. P-450) inducible under conditions of glucose repression. Under these conditions the enzyme has maximal expression of 0.43 nmol g^?1 wet wt at the end of the exponential phase of growth. Substrate and inhibitor affinity was examined using studies of spectral changes on binding and revealed a type II spectrum with ketoconazole (K_s = 23 μM ) and a type I spectrum with benzo(a)pyrene (K_s = 77 μM ). A K_m of 112 μM was found in the aryl hydrocarbon hydroxylas assay. These properties show broad comparability with the cyt. P-450 of Saccharomyces cerevisiae.     

Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe.

The architectural features of 73 introns found in 36 genes of the fission yeast Schizosaccharomyces pombe have been compiled and tabulated. The introns from S. pombe can be grouped into two size classes. Intron features are discussed in comparison to intron features of Saccharomyces cerevisiae and other eukaryotes. The results indicate that S. pombe displays quite different architectural features than the budding yeast S. cerevisiae. However, particularly in the 3' region, S. pombe introns also appear to differ from mammalian introns.     

Homologous chromosome pairing in Schizosaccharomyces pombe

Homologous chromosome pairing is a central feature of meiosis I, contributing to the correct segregation of chromosomes during meiosis. The fission yeast, Schizosaccharomyces pombe, has been widely used to study meiotic chromosome dynamics, partly because studies in this yeast are simplified due to the lack of post-pairing synaptic structures. Chromosome pairing in Sz. pombe occurs differentially throughout the genome. Telomeres cluster at the spindle pole body (SPB) at the onset of meiosis, imposing a spatial restriction on pairing events. Subsequently, centromeres dissociate from the SPB and pair in a recombination- and heterochromatin (Swi6)-independent fashion. Pairing of telomere distal regions occurs during meiotic prophase, concomitant with a dynamic association/dissociation of homologous regions, with interhomologue associations becoming increasingly stable. The stabilization of paired regions is enhanced by factors required for the initiation of meiotic recombination, suggesting that recombination stabilizes paired regions. However, substantial pairing is initiated in the absence of recombination; this is dependent upon another factor, the conserved Meu13 protein, demonstrating that recombination is not required for initial pairing interactions. During meiotic prophase Sz. pombe exhibits a pronounced dynein-dependent nuclear oscillation, which drives the pairing of centromeric and interstitial regions. Dynein is also required for the significant levels of achiasmate reductional segregation observed in Sz. pombe, possibly implicating the centromere-associated pairing with achiasmate homologue segregation. Whilst Sz. pombe does not form discernable synaptic structures continuously along the meiotic chromosomes, it does form proteinacious, meiosis-specific, linear structures (linear elements). However, the role, if any, of these structures in mediating homologue pairing is unknown.     

Transport of L-leucine in the fission yeast Schizosaccharomyces pombe

Transport of L -leucine into Schizosaccharomyces pombe cells from the stationary phase of growth (after preincubation for 60 min with 1% glucose) proceeds uphill, practically unidirectionally, and is mediated by at least two systems: a high-affinity system with a K_T of 0·045 mmol 1^?1 and J_max of 3·3 nmol min^?1 (mg dry weight)^?1 and a low-affinity system with a K_T of 1·25 mmol 1^?1 and J_max of 16·0 nmol min^?1 (mg dry weight)^?1. The high-affinity system has a pH optimum at 3.2, the accumulation ratio is highest at a cell density of 2–4 mg dry weight per ml and decreases with increasing leucine concentration. Transport of leucine by the high-affinity system is strongly inhibited by proton conductors, ammonium ions and by most amino acids, but only L -phenylalanine, L -isoleucine, L -valine and L -cysteine behave as fully competitive inhibitors. Systems of L -leucine transport in S. pombe are not constitutive. Transport activity appears only after preincubation of cells with a suitable source of energy. If cycloheximide is added during preincubation with glucose, no transport systems for leucine are synthesized. After removal of glucose, the activity of transport systems decays with a half-time of about 20 min. The presence of cyclic AMP increases the initial rate of leucine uptake only in cells preincubated with glucose and in the absence of cycloheximide.     

Sterility (ste) genes of Schizosaccharomyces pombe

In previous experiments of Girgsdies (1982), eight sterile (ste) mutants of Schizosaccharomyces pombe did not sporulate when fused with h⁺ or h^? protoplasts. We succeeded in achieving sporulation with these mutants. Two hitherto unknown ste genes, ste7 and ste8, were found.     

Interpretations of ultraviolet absorption in white wines

Methods are described for the general analysis of total ultraviolet (u.v.) absorbing constituents of white wines. An elution profile, which is characteristic of the particular wine, is obtained by continuous monitoring of effluent from a dextran gel column at 280 nm. Surprisingly, about half of the total u.v. absorbance in the Riesling wine examined was found to be due to non-phenolic wine constituents, which are separately eluted in the gel column analysis. These are nucleotidic materials, to which little attention has previously been given in oenology. Data relating to the protein-tannin fraction, and to nucleotidic and phenolic constituents, are directly obtained by recording the wine elution profiles at 265, 280 and 320 nm. The interpretation of the elution curves and the significance and general utility of this new analytical method are discussed.     

Isolation and characterization of Schizosaccharomyces pombe fragile mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.     

利用粟酒裂殖酵母生长法定量测定肌醇浓度

利用本实验室分离得到的一株天然的肌醇合成缺陷型的粟酒裂殖酵母 (Schizosaccha romycespombe)特异地依赖外部肌醇生长的特性 ,研究了该菌株在含有不同浓度肌醇的合成培养基中的生长情况 ,发现在一定的肌醇浓度范围内 ,菌株的生物量随肌醇浓度增大而增大 ,且呈线性关系 ,由此建立了一套微生物法定量测定肌醇浓度的方法。我们发现在固定的培养温度、振荡速度等外部条件下 ,当接种量OD60 0 为 1 0、葡萄糖浓度为 4 0 %时 ,在肌醇浓度 0～ 5mg/L范围内 ,培养 52h后 ,肌醇测定标准曲线最为理想 ,R值为 0 9973。应用该方法对标准肌醇样品的测定得到了较好的结果     

Volatile components of Zalema white wines

The volatile composition of young white wines from Vitis vinifera cv. Zalema, an autochthonous grape variety in Huelva (southern Spain), has been studied by gas chromatography-olfactometry (GC-O) and techniques of quantitative analysis. This is the first time that an olfactometric analysis has been reported in wines made from this grape variety. The quantitative chemical study has shown 71 volatile compounds, of which 23 were in concentrations above their thresholds. On the basis of the odour activity values (OAVs), the most potent odorants were fermentative compounds, mainly fatty acids and their ethyl esters. Two norisoprenoids, β-damascenone and β-ionone, two alcohols (isoamyl alcohol and β-phenylethanol), three volatile thiols, 4-mercapto-4-methyl-2-pentanone, 3-mercaptohexyl acetate and 3-mercapto-1-hexanol, and two carbonyl compounds (acetaldehyde and phenylacetaldehyde) also exhibited OAVs > 1. The GC-O study corroborated these results, showing that five esters (isoamyl acetate, ethyl hexanoate, ethyl butyrate, ethyl isovalerate and ethyl octanoate), isoamyl alcohol and β-damascenone can be considered as the most powerful odorants of Zalema wines.     

Double-strand break repair and homologous recombination in Schizosaccharomyces pombe

The study of double-strand break repair and homologous recombination in Saccharomyces cerevisiae meiosis has provided important information about the mechanisms involved. However, it has become clear that the resulting recombination models are only partially applicable to repair in mitotic cells, where crossover formation is suppressed. In recent years our understanding of double-strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organism for double-strand break repair. In this review we will focus on the involvement of homologous recombination and repair in different aspects of genome stability in Sz. pombe meiosis, replication and telomere maintenance. We will also discuss anti-recombination pathways (that suppress crossover formation), non-homologous end-joining, single-strand annealing and factors that influence the choice and prevalence of the different repair pathways in Sz. pombe.     

Purification and characterization of aminopeptidase yspI from Schizosaccharomyces pombe

Aminopeptidase yspI was purified to apparent homogeneity from the fission yeast Schizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl-4-nitroanilides is 7·0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L-lysine-4-nitroanilide with high efficiency.     

Yeasts used to delay browning in white wines

Commercial white wines of the Sherry type were subjected to accelerated browning at 35 °C in the absence and presence of yeasts at concentrations of 1, 1.5 and 2 g/l. Based on the results, the yeasts delayed browning in the wines, measured in terms of the absorbance at 420 nm, the effect increasing with increase in the yeast concentration. The addition of yeasts was also found to affect phenolic compounds, particularly decreasing the concentrations of brown compounds. Wines of the same type stored in stoppered bottles at 19 °C for 12 months exhibited a considerable delay of browning in the presence of yeasts with not-too-serious alteration of their sensory properties, which made them still acceptable for consumption.     

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.     

Characterization of two fructosyl-amino acid oxidase homologs of Schizosaccharomyces pombe

Two putative fructosyl-amino acid oxidase genes, FAP1 and FAP2, found in the Schizosaccharomyces pombe genome were cloned and expressed. Both of the gene products (Fap1 and Fap2) were flavoproteins and have no activity for fructosyl-amino acids. It was suggested that Fap1 and Fap2 are an L-pipecolic acid oxidase and L-saccharopine oxidase, respectively.     

Pro-oxidant action of phloxine B on fission yeast Schizosaccharomyces pombe

A Schizosaccharomyces pombe mutant deficient in Cu,Zn-superoxide dismutase (sod1 mutant) was hypersensitive to phloxine B, which is used as a food-colouring agent and also to distinguish diploid strains of Sz. pombe from haploid strains, under illumination with light. The pro-oxidant nature of phloxine B was confirmed biochemically. The carbonyl content of proteins (which represents protein oxidation) increased, and the reduced form of glutathione was transiently decreased by phloxine B treatment under illumination with light. When cells were treated with phloxine B under light, carbonyl content of proteins in the sod1 mutant was greater than that in the wild-type and amount of glutathione was much decreased in the sod1 mutant compared with the wild-type. Genes induced by oxidative stress were induced by phloxine B under illumination with light and some were induced by phloxine B without light.     

Chemical synthesis of the M-factor mating pheromone from Schizosaccharomyces pombe

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones. We recently showed that M-factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C-terminal cysteine residue is carboxyl-methylated and S-alkylated, probably with a farnesyl residue (Davey, 1992): Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)-OCH₃. Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone. These results confirm the structure of the M-factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast.     

Expression of two Trichoderma reesei xylanases in the fission yeast Schizosaccharomyces pombe

Two xylanase genes (xyn1 and xyn2) were amplified by the polymerase chain reaction (PCR) technique from first-strand cDNA prepared from mRNA of Trichoderma reesei QM9414. The genes were located under the human cytomegalovirus gene promoter (CMVp) on copy-number-controlled plasmids (pTLxyn1 and pTLxyn2). When both plasmids were introduced into Schizosaccharomyces pombe, functional xylanases (XYN I and XYN II) were secreted by the recombinant yeasts. The secreted XYN I protein had a molecular mass of 21 kDa whereas XYN II was produced as two molecular forms with sizes of 21 and 28 kDa, the former being not glycosylated and the latter N-glycosylated. XYN I was secreted in the culture medium at a level of about 25 microg/ml and XYN II at about 170 microg/ml. The recombinant xylanases had the same characteristics with respect to the effects of temperature and pH on the enzyme activity as the native ones.     

Volatile sulphur compounds composition of monovarietal white wines

The sulphur compounds composition of wines produced experimentally from six white cultivars (Alvarinho, Loureiro, Trajadura, Pedernã, Azal Branco and Avesso) was evaluated during two consecutive vintages. Results show that wines could be differentiated according to their sulphur compounds content. In general, Loureiro, Trajadura and Pedernã cultivars led to wines with low concentrations of sulphur compounds; however, Loureiro wines were characterised by significant amounts of dimethyl sulphone, whereas Trajadura wines possessed a high content of 3-(methylthio)propyl acetate and 4-(methylthio)-1-butanol. Alvarinho and Avesso wines showed high levels of S-methyl thioacetate, 3-mercapto-1-propanol, 3-(ethylthio)-1-propanol and 3-methylthiopropionic acid. Significant amounts of 2-methyltetrahydrothiophen-3-one, cis- and trans-2-methyltetrahydrothiophen-3-ol were also found in Avesso wines. Azal Branco wines were low in 3-(methylthio)propyl acetate and 2-(methylthio)ethanol, and high contents in S-methyl thioacetate, 3-mercapto-1-propanol and 2-mercaptoethanol. A linear discriminant analysis of sulphur compounds levels showed a differentiation of wines according to their varietal origin.