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Crosses between Saccharomyces cerevisiae and Saccharomyces bayanus generate fertile hybrids.

Crossings between strains of Saccharomyces cerevisiae and Saccharomyces bayanus were carried out. Genetic, molecular and electrophoretic karyotyping data indicated that interspecific hybrids were obtained. The hybrid cells segregated "grande" and "petite" colonies, and the latter ranged between 20 and 50%; unlike "grande" colonies, "petite" colonies did not sporulate and did not ferment maltose. In the hybrids, the extent of sporulation varied between 10 and 20%; only very rare asci (around 10(-4)) held viable ascospores. Clones from the viable ascospores sporulated and produced asci with viable ascospores able to give mating with spores from both hybrid derivatives and parental species. Fertile asci could derive from allotetraploid cells generated by endomitotic events in allodiploid cells, a mechanism that enables overcoming the species barrier between S. cerevisiae and S. bayanus.

Crosses, Genetic↗

Genetic variation of the repeated MAL loci in natural populations of Saccharomyces cerevisiae and Saccharomyces paradoxus.

In Saccharomyces cerevisiae, the gene functions required to ferment the disaccharide maltose are encoded by the MAL loci. Any one of five highly sequence homologous MAL loci identified in various S. cerevisiae strains (called MAL1, 2, 3, 4 and 6) is sufficient to ferment maltose. Each is a complex of three genes encoding maltose permease, maltase and a transcription activator. This family of loci maps to telomere-linked positions on different chromosomes and most natural strains contain more than one MAL locus. A number of naturally occurring, mutant alleles of MAL1 and MAL3 have been characterized which lack one or more of the gene functions encoded by the fully functional MAL loci. Loss of these gene functions appears to have resulted from mutation and/or rearrangement within the locus. Studies to date concentrated on the standard maltose fermenting strains of S. cerevisiae available from the Berkeley Yeast Stock Center collection. In this report we extend our genetic analysis of the MAL loci to a number of maltose fermenting and nonfermenting natural strains of S. cerevisiae and Saccharomyces paradoxus. No new MAL loci were discovered but several new mutant alleles of MAL1 were identified. The evolution of this gene family is discussed.

Alleles↗

Saccharomyces Genome Database (SGD) provides tools to identify and analyze sequences from Saccharomyces cerevisiae and related sequences from other organisms.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/), a scientific database of the molecular biology and genetics of the yeast Saccharomyces cerevisiae, has recently developed several new resources that allow the comparison and integration of information on a genome-wide scale, enabling the user not only to find detailed information about individual genes, but also to make connections across groups of genes with common features and across different species. The Fungal Alignment Viewer displays alignments of sequences from multiple fungal genomes, while the Sequence Similarity Query tool displays PSI-BLAST alignments of each S.cerevisiae protein with similar proteins from any species whose sequences are contained in the non-redundant (nr) protein data set at NCBI. The Yeast Biochemical Pathways tool integrates groups of genes by their common roles in metabolism and displays the metabolic pathways in a graphical form. Finally, the Find Chromosomal Features search interface provides a versatile tool for querying multiple types of information in SGD.

Amino Acid Sequence↗

Genome Snapshot: a new resource at the Saccharomyces Genome Database (SGD) presenting an overview of the Saccharomyces cerevisiae genome.

Sequencing and annotation of the entire Saccharomyces cerevisiae genome has made it possible to gain a genome-wide perspective on yeast genes and gene products. To make this information available on an ongoing basis, the Saccharomyces Genome Database (SGD) (http://www.yeastgenome.org/) has created the Genome Snapshot (http://db.yeastgenome.org/cgi-bin/genomeSnapShot.pl). The Genome Snapshot summarizes the current state of knowledge about the genes and chromosomal features of S.cerevisiae. The information is organized into two categories: (i) number of each type of chromosomal feature annotated in the genome and (ii) number and distribution of genes annotated to Gene Ontology terms. Detailed lists are accessible through SGD's Advanced Search tool (http://db.yeastgenome.org/cgi-bin/search/featureSearch), and all the data presented on this page are available from the SGD ftp site (ftp://ftp.yeastgenome.org/yeast/).

Chromosomes, Fungal↗

On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.

Cell Wall↗

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR.

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

DNA, Fungal↗

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae.

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.

Amino Acid Sequence↗

Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae.

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

Animals↗

Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions.

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.

Acetaldehyde↗

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.

Cloning, Molecular↗

Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH.

The yeast assimilatory sulfate reductase is a complex enzyme that is responsible for conversion of sulfite into sulfide. To obtain information on the nature of this enzyme, we isolated and sequenced the MET10 gene of Saccharomyces cerevisiae and a divergent MET10 allele from Saccharomyces carlsbergensis. The polypeptides deduced from the identically sized open reading frames (1,035 amino acids) of both MET10 genes have molecular masses of around 115 kDa and are 88% identical to each other. The transcript of S. cerevisiae MET10 has a size comparable to that of the open reading frame and is transcriptionally repressed by methionine in a way similar to that seen for other MET genes of S. cerevisiae. Distinct homology was found between the putative MET10-encoded polypeptide and flavin-interacting parts of the sulfite reductase flavoprotein subunit (encoded by cysJ) from Escherichia coli and several other flavoproteins. A significant N-terminal homology to pyruvate flavodoxin oxidoreductase (encoded by nifJ) from Klebsiella pneumoniae, together with a lack of obvious flavin mononucleotide-binding motifs in the MET10 deduced amino acid sequence, suggests that the yeast assimilatory sulfite reductase is a distinct type of sulfite reductase.

Amino Acid Sequence↗

Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist.

Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.

Amino Acid Sequence↗

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii.

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.

DNA, Fungal↗

The nucleotide sequences of the actin genes from Saccharomyces carlsbergensis and Saccharomyces cerevisiae are identical except for their introns.

The actin gene from yeast Saccharomyces carlsbergensis was cloned in Escherichia coli and its complete nucleotide structure was determined. A comparison of its DNA sequence with that of the related yeast species Saccharomyces cerevisiae revealed that the coding as well as the 5'- and 3'-untranslated regions are identical. The intron, although at the same location in the two genes, differs in three positions. There is one deletion (or insertion), one transition, and one transversion. These are clustered within and around an oligo(dA) stretch of 14 (or 13) residues. Our observations identify the intron as the fastest evolving segment of this eukaryotic gene.

Actins↗

Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae.

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).

Acetone↗

In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains.

The probiotic potential of 18 Saccharomyces cerevisiae strains used for production of foods or beverages or isolated from such, and eight strains of Saccharomyces cerevisiae var. boulardii, was investigated. All strains included were able to withstand pH 2.5 and 0.3% Oxgall. Adhesion to the nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of 3H-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13.6-16.8%) recorded for two isolates from blue veined cheeses. Merely 25% of the S. cerevisiae var. boulardii strains displayed good adhesive properties (16.2-28.0%). The expression of the proinflammatory cytokine IL-1alpha decreased strikingly in IPEC-J2 cells exposed to a Shiga-like toxin 2e producing Escherichia coli strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness.

Animals↗

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation.

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.

Cell Proliferation↗

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome.

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.

Beer↗