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At least 271 records · Page 15Linked to original sources
PubMed1997

Multidrug-resistant transport proteins in yeast: complete inventory and phylogenetic characterization of yeast open reading frames with the major facilitator superfamily.

Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae reveals that 28 open reading frames (ORFs) are homologous to each other and to established bacterial members of the drug-resistant subfamily of the major facilitator superfamily. The phylogenesis of these protein sequences shows that they fall into three major clusters. Cluster I contains 12 ORFs, cluster II contains ten ORFs and cluster III contains six ORFs. Hydropathy analyses indicate that in cluster II and III ORFs, 14 transmembrane spans are predicted whereas only 12 transmembrane spans are predicted in cluster I ORFs. Three ORFs that have known functions as multidrug-resistance pumps in other yeast species such as Schizosaccharomyces pombe (CAR1), Candida albicans (BMRP) or C. maltosa (CYHR), also fall into cluster I. Two S. cerevisiae ORFs of known multidrug-resistance function (ATR1, SGE1) fall into cluster II. Cluster III consists exclusively of ORFs of unknown function but binary sequence comparisons show homology to ORFs from cluster II. Analysis of the multiple alignment for these proteins leads to the identification of characteristic signature sequences for each of the three clusters.

Amino Acid Sequence↗
PubMed2000

Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras.

The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling.

GTP-Binding Proteins↗
PubMed2004

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes.

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.

Cluster Analysis↗
PubMedDate not supplied

Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase.

Kinetic study of the mechanism of the temperature-induced loss of the catalytic activity by yeast hexokinase (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDG) has shown the dissociative nature of the processes. In the temperature range 40-47 degrees C, they are satisfactorily described in terms of consecutive reactions in which steps of irreversible denaturation of the monomeric units follow the reversible dissociation of inactive oligomeric forms into the active units, resulting in an increase in catalytic activity. The experimental data have been analyzed in the framework of the dissociative mechanism, and a semiquantitative method has been developed for calculating the individual rate constants.

Enzyme Stability↗
PubMed1996

Production of extracellular beta-mannanases by yeasts and yeast-like microorganisms.

A new screening method for simultaneous detection of endo-beta-1,4-mannanase and endo-beta-1,4-xylanase producing microorganisms is described. Two differently dyed substrates Ostazin Brilliant Red-galactomannan and Remazol Brilliant Blue-xylan were incorporated into the same agar media. Decolorizing of one or both substrates around the cell colonies indicates secretion of the corresponding enzyme(s). The method was used to screen 449 yeasts and yeast-like microorganisms belonging to 68 different genera. The secretion of endo-beta-1,4-mannanases and/or endo-beta-1,4-xylanases was found within 10 genera (42 positive strains out of 261 tested). A low frequency of occurrence of endo-beta-1,4-mannanases was observed within the genera Cryptococcus (1 positive strain out of 15 tested), Geotrichum (1 of 6) and Pichia (1 of 35). The highest frequency of occurrence of endo-beta-1,4-mannanases was found within the genera Stephanoascus (2 of 2) and Aureobasidium (14 of 14). Strains hydrolyzing Ostazin Brilliant Red-galactomannan were cultivated in liquid media containing 1% locust bean gum. The best producers of extracellualr endo-beta-1,4-mannanases were found to be the strains of Aureobasidium pullulans.

Bacteriological Techniques↗
PubMed1981

Anomeric specificity of the monosaccharide carrier in yeasts and yeast-like organisms.

The anomeric specificity of monosaccharide uptake was investigated in 42 species of yeasts and related mycelium-forming fungi. Differences in the uptake of anomers were determined by the following methods. (1) Shift of anomeric equilibrium in the outer medium caused by preferential uptake of one of the anomeric forms was monitored polarimetrically as induced mutarotation. (2) The uptake of 14C-D-glucose by cells was examined after addition of freshly prepared solutions of alpha- or beta-D-glucose. Most of the organisms examined display the Saccharomyces-type preference for the alpha-anomers of glucose and xylose which is caused by the higher affinity of the monosaccharide carrier for the alpha-pyranose configuration. The following genera show this type of preference (the number of species is given in parenthesis): Saccharomyces (5), Schizosaccharomyces (1), Endomycopsis (2), Eremascus (1), Endomyces (1), Pichia (1), Hansenula (1), Debaryomyces (2), Lipomyces (1), Willia (1), Nematospora (1), Kluyveromyces (2), Candida (5), Torulopsis (5), Cryptococcus (1). No anomeric specificity was shown by the following genera: Nadsonia (1), Dipodascus (2), Rhodotorula (5), Sporobolomyces (2), Bullera (1), Rhodosporidium (1). A parallel investigation of the concentration dependence of glucose uptake indicates that most yeasts possess a constitutive monosaccharide carrier characterized by the following features: a high maximum rate of uptake, a relatively low affinity, and preference for alpha-anomers. Besides this carrier the majority of these microorganisms possess a glucose-transporting carrier with a higher affinity and a lower capacity.

Biological Transport, Active↗
PubMed1998

Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA.

In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit. L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay. In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture. Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one. Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner. Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA. However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule. L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4x10(6)/M. L17 bound the pre-rRNA molecule preferentially (Ka=17x10(6)/M) but also bound the mature 25S rRNA species (Ka=10x10(6)/M). L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well (Ka=11-12x10(6)/M). L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with Ka=3.1 vs. 2.5x10(6)M, respectively. We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.

Binding, Competitive↗
PubMed1999

Functional interaction of yeast elongation factor 3 with yeast ribosomes.

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.

Adenosine Triphosphatases↗
PubMed2001

The seasonal variability of yeasts and yeast-like organisms in water and bottom sediment of the Szczecin Lagoon.

Qualitative and quantitative analysis of samples of water and bottom sediment from the Szczecin Lagoon were carried out from May, 1996 to December, 1997. Isolates of yeasts and yeast-like fungi from water samples reached maximum counts in July whereas sediment samples revealed highest counts in May. Qualitative analysis showed that Candida famata and Rhodothorula rubra were dominant species. As they are typical of strongly eutrophic water their presence indicates that the Szczecin Lagoon is significantly polluted by industrial and municipal sewage.

Candida↗
PubMed1999

Multicenter comparison of the sensititre YeastOne Colorimetric Antifungal Panel with the National Committee for Clinical Laboratory standards M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeasts and yeast-like organisms.

National Committee for Clinical Laboratory Standards (NCCLS) standard guidelines are available for the antifungal susceptibility testing of common Candida spp. and Cryptococcus neoformans, but NCCLS methods may not be the most efficient and convenient procedures for use in the clinical laboratory. MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined by the commercially prepared Sensititre YeastOne Colorimetric Antifungal Panel and by the NCCLS M27-A broth microdilution method for 1,176 clinical isolates of yeasts and yeast-like organisms, including Blastoschizomyces capitatus, Cryptococcus spp., 14 common and emerging species of Candida, Hansenula anomala, Rhodotorula spp., Saccharomyces cerevisiae, Sporobolomyces salmonicolor, and Trichosporon beigelii. Colorimetric MICs of amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first purple or blue well. Three comparisons of MIC pairs by the two methods were evaluated to obtain percentages of agreement: 24- and 48-h MICs and 24-h colorimetric versus 48-h reference MICs. The best performance of the YeastOne panel was with 24-h MICs (92 to 100%) with the azoles and flucytosine for all the species tested, with the exception of C. albicans (87 to 90%). For amphotericin B, the best agreement between the methods was with 48-h MIC pairs (92 to 99%) for most of the species tested. The exception was for isolates of C. neoformans (76%). These data suggest the potential value of the YeastOne panel for use in the clinical laboratory.

Amphotericin B↗
PubMed2006

A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones.

The yeast artificial chromosomes (YAC) system makes it possible not only to clone large DNA fragments but also to simplify the physical mapping and functional analysis of chromosomes and genomes through YAC manipulation. To manipulate large DNA fragments cloned into YACs, YAC fragmentation has been developed and also a new method called the YAC-splitting method was developed recently. Although the YAC fragmentation method can be used to delete DNA from one side of a YAC, the YAC-splitting method generates two smaller YACs, both of which are transmitted to daughter cells during mitotic growth. This chapter describes the YAC-splitting method improved by incorporating polymerase chain reaction-mediated chromosome splitting (PCS) technique and by adding yeast autonomously replicating sequence (ARS) to the system. The PCS method combines a streamlined procedure (two-step PCR and one transformation per splitting event) with the Cre/loxP system for marker rescue. The improved YAC-splitting method can convert a targeted region of a eukaryotic chromosome within a YAC into a new replicating YAC.

Chromosomes, Artificial, Yeast↗
PubMed1982

Light effects in yeast: relation between the respiratory deficiency and light sensitivity in yeast.

Visible light of 5,000 lux intensity has been shown to photokill yeast cells at 12 degrees C. In the present report some of isogenic respiratory deficient mit- and nuclear mutants were compared for their sensitivity to light. No close correlation between the cytochromes spectra and light resistance was observed. Although, the nuclear and rho- mutants which lack cytochromes a + a3 and b are as a rule light resistant. Photokilling effect in yeast seems to be dependent both on the sufficiency of respiratory chain and on protein synthesis probably on cytoplasmic level.

Culture Techniques↗
PubMed1995

Seven megabase yeast artificial chromosome contig at region 11p15: identification of a yeast artificial chromosome spanning the breakpoint of a chromosomal translocation found in a case of Beckwith-Wiedemann syndrome.

Genetic alterations of chromosome region 11p15 have been detected in neoplastic diseases as well as in cancer-predisposing syndromes. The cloning of the entire chromosomal region will be important for the identification and characterization of critical tumor suppressor genes. We have developed a yeast artificial chromosome contig that covers up to 7 Mb of this chromosome band. The most centromeric marker included in the contig is D11S932 and the most telomeric is D11S470. We have developed 18 new STS markers, which have been located in the contig in relation to 16 known markers. One of the yeast artificial chromosome clones was found to span the chromosome 11 breakpoint of the translocation t(11;18), associated with a case of Beckwith-Wiedemann syndrome. Cloning the regions in proximity to this translocation might reveal the presence of a gene altered in association with the development of Beckwith-Wiedemann syndrome.

Base Sequence↗
PubMed1996

Sequencing of a 17.6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs, including CCT, ADE3 and TR-I genes, homologues of the yeast PMT and EF1G genes, of the human and bacterial electron-transferring flavoproteins (beta-chain) and of the Escherichia coli phosphoserine phosphohydrolase, and five new ORFs.

A 17.6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1gamma, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (beta chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.

Amino Acid Sequence↗
PubMed1999

Mouse CAF1, a mouse homologue of the yeast POP2 gene, complements the yeast pop2 null mutation.

The yeast POP2 protein (Pop2p) is a component of a global transcription regulatory complex and is required for gene expression of many genes in Saccharomyces cerevisiae. We constructed POP2 deletion plasmids encoding various Pop2p regions under the native POP2 promoter and found that the minimum functional region was located in two-thirds of the carboxyl terminal region. A mouse homologue of the POP2 gene (mCAF1), which corresponds to the Pop2p minimum region, partially rescued the growth defect of pop2 null mutant cells. Addition of the Pop2p amino terminal region to mCAF1 strengthened the suppression. mCAF1 also weakly suppressed the relatively high expression of the SUC2 gene of pop2 cells under glucose-repressing conditions; however, it failed to suppress the defect of full expression of the SUC2 gene under glucose-derepressing conditions. Our findings clearly demonstrate that a mammalian homologue can substitute for the yeast POP2 gene in some aspect.

Animals↗
PubMed2000

Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm.

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

Animals↗