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At least 199 records · Page 11Linked to original sources
PubMed1992

Dimorphism in Itersonilia perplexans: yeast and hyphal phases differ in their sensitivity to mycocins produced by tremellaceous yeasts.

The monokaryotic yeast phase of the heterobasidiomycete Itersonilia perplexans, unlike the hyphal phase, was found to be sensitive to mycocins produced by killer strains of Cryptococcus humicola, Cr. laurentii, Cystofilobasidium bisporidii and Rhodotorula fujisanense. Both the yeast and hyphal phases wer resistant to mycocins of Cr. podzolicus, Filobasidium capsuligenum, Rhodotorula glutinis, Rh. mucilaginosa, Rh. pallida, Sporidiobolus johnsonii, Sb. pararoseus and Sporobolomyces alborubescens. The different sensitivity patterns of yeast and hyphal phases are probably caused by biochemical differences in the cell walls.

Cell Wall↗
PubMed1994

High-throughput rapid yeast DNA extraction. Application to yeast artificial chromosomes as polymerase chain reaction templates.

An automation-assisted method enables DNA extraction of over a thousand yeast colonies in a day by one person. Yeast DNA is extracted essentially quantitatively in three steps: (a) cells are converted into spheroplasts with yeast lytic enzyme, (b) cells are lysed with proteinase K, and (c) DNA is collected by ethanol precipitation. The DNA stored at 4 degrees C is active in polymerase chain reaction experiments for more than a year.

Chromosomes, Artificial, Yeast↗
PubMed1994

Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.

Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.

Amino Acids↗
PubMed1999

A mathematical model for toxin accumulation by killer yeasts based on the yeast population growth.

The accumulation of toxin by killer yeast populations is modelled starting from a mechanistic approach that explains the toxin production in terms of yeast population growth, and takes into account the environmental inactivation of the toxin. A modified Richard's general equation for limited growth is used to define the function that describes the toxin produced in relation to the yeast biomass increase. The relationship between the rates of cell and toxin production is explicitly shown, and the implications of the resulting proportionality factor are discussed. The model parameters have been adjusted and the model has been validated using experimental data of growth and toxin accumulation from cultures of Pichia membranaefaciens in two different media. The differences between both types of cultures are analysed on the basis of parameter estimates and the predicted rate of toxin production per cell. The results support the hypothesis that biomass production and toxin synthesis are controlled in different ways; they also suggest that the composition of the medium could have a distinct effect on toxin synthesis. Model assumptions are discussed in comparison with a previous model for killer-sensitive interaction of Saccharomyces cerevisiae strains.

Computer Simulation↗
PubMed1990

Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells.

Cell Membrane↗
PubMed1994

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.

Amino Acid Sequence↗
PubMed1975

Three yeast proteins that specifically inhibit yeast proteases A, B, and C.

Baker's yeast was found to contain inhibitors of yeast proteases A and C. These two proteins were partially purified, characterized, and compared with the previously described inhibitor of protease B. The A and B inhibitors were very thermostable and were extracted from intact yeast cells at 9k C. The A inhibitor appeared to be a protein with a molecular weight of about 22,000 which could be dissociated into two monomers or chains, both of which had a molecular weight of approximately 11,000. The protease C (carboxypeptidase Y)-inhibitor complex was purified and then partially disociated on an ion-exchange column. The free protease C inhibitor was very unstable, possibly because of destruction by a contaminating protease. Each inhibitor was specific for its corresponding protease and each inhibition was competitive. Whereas proteases A, B, and C destroyed the B inhibitor, only protease B had a pronounced destructive effect on the protease A inhibitor. Pepstatin was found to be a selective inhibitor of protease A, whereas chymostatin and antipain specifically inhibited protease B.

Carboxypeptidases↗
PubMed1975

Evaluation of the Uni-Yeast-Tek kit for the identification of medically important yeasts.

The Uni-Yeast-Tek system, a commercially prepared kit and scheme for the rapid identification of medically important yeasts (Corning Medical), was evaluated in comparison with a conventional procedure in the identification of 623 yeasts. The system permitted the presumptive identification of 99.8% of 436 isolates representing 16 common species commonly isolated in the clinical laboratory. Correct biochemical and morphological analyses were obtained with 48 other species, but their specific identification required additional data.

Candida↗
PubMed2005

Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin.

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.

Actins↗
PubMed1998

[Studies on the yeasts and yeast-like fungi degrading trinitrotoluene].

Seventeen strains of yeasts and yeast-like fungi were isolated from soil and waste water samples polluted by trinitrotoluene (TNT). These strains can degrade 71%-93% TNT when growing in the medium originally containing 70-80 mg/L TNT within 40 h. They were identified. Among them, six strains are Candida krusei, four are C. quercitrusa, one is C. famata, one is Hansenula beijerinckii, one is H. subpelliculosa, and four are Geotrichum candidum. Six strains were selected for further studies on the conditions effecting TNT degradation by them. The optimum pH and temperature are pH 7 and 37-40 degrees C, respectively. TNT Degradation ability of the strains can be promoted by adding 0.01%-0.05% glucose or 0.01%-0.1% yeast extract into the medium. Addition of 0.05% (NH4)2SO4 or NH4Cl to the medium can evidently inhibit the degradation of TNT by the strains.

Biodegradation, Environmental↗
PubMed2004

[Construction of plasmid with green fluorescent protein gene for analysis of yeasts in yeast wastewater treatment].

Green fluorescent protein (GFP) can be utilized in analysis of the characteristics and distribution of a targeted strain in microbial communities. This study is the first step to establish a dynamic yeast monitoring technique in a wastewater treatment system using yeast by constructing a fluorescent yeast containing gfp gene. The gfp gene was inserted into pACT1-URA3, a powerful plasmid for introducing a foreign gene into Candida boidinii, and then transformed into E. coli JM109. The gfp gene was expressed, though not very highly. The results of the electrophoresis and polymerase chain reaction suggested that the newly constructed plasmid containing gfp gene might not exist in free form in the cells, but in some special way such as interaction with the chromosome.

Candida albicans↗
PubMed1983

Homologous nucleotide sequences at the 5' termini of messenger RNAs synthesized from the yeast enolase and glyceraldehyde-3-phosphate dehydrogenase gene families. The primary structure of a third yeast glyceraldehyde-3-phosphate dehydrogenase gene.

Genomic DNA containing a third yeast glyceraldehyde-3-phosphate dehydrogenase structural gene has been isolated on a bacterial plasmid designated pgap11. The complete nucleotide sequence of this structural gene was determined. The gene contains no intervening sequences, codon usage is highly biased, and the nucleotide sequence of the coding portion of this gene is 90% homologous to the other two glyceraldehyde-3-phosphate dehydrogenase genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). Based on the extent of nucleotide sequence divergence among the three glyceraldehyde-3-phosphate dehydrogenase genes, it is likely that they arose as a consequence of two duplication events and the gene contained on the hybrid plasmid designated pgap11 is a product of the first duplication event. All three structural genes share extensive nucleotide sequence homology in the 5'-noncoding regions adjacent to the three respective translational initiation codons. The gene contained on pgap11 is not homologous to the others downstream from the respective translational termination codon, however. The 5' termini of messenger RNAs synthesized from the three glyceraldehyde-3-phosphate dehydrogenase and two yeast enolase genes have been mapped to sites ranging from 36 to 82 nucleotides upstream from the respective translational initiation codons. In each case the 5' terminus of the mRNA maps to a region of strong nucleotide sequence homology which is shared by all five structural genes. These latter data confirm that all five structural genes are expressed during vegetative cell growth and further support the hypothesis that a portion of the 5'-noncoding flanking region of the yeast glyceraldehyde-3-phosphate dehydrogenase and enolase genes evolved from a common precursor sequence.

Base Sequence↗
PubMed1994

Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide.

We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme. The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations. The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations. The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity. We describe three mutants that we isolated using this system. Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine. DNA sequence changes have been identified in all of these mutant TOP2 genes. The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria. One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2. Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

Amino Acid Sequence↗
PubMed2003

Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process.

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.

Adenine↗
PubMed1986

Transcription and expression of zein sequences in yeast under natural plant or yeast promoters.

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23-kd protein have been inserted in an interspecific Escherichia coli-Saccharomyces cerevisiae expression vector in different constructions. The presence of the inducible GAL1-10 upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC-1 promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC-1 gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.

Journal Article↗
PubMed1992

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.

Amino Acid Sequence↗
PubMed1995

A yeast transcription factor bypassing the requirement for SBF and DSC1/MBF in budding yeast has homology to bacterial signal transduction proteins.

The transcription factors SBF and DSC1/MBF bind SCB and MCB promoter elements, respectively, and are essential for the cell cycle progression of Saccharomyces cerevisiae through the control of G1 cyclin gene expression. We isolated a gene (BRY1; Bacterial Response regulator in Yeast) able to activate either MCB or SCB promoter elements on a reporter plasmid which, when overexpressed, can bypass the normally essential requirement for SBF and DSC1/MBF by the stimulation of CLN1 and CLN2 expression. In the case of CLN2 at least, this expression depends upon the MCB and SCB promoter elements. In wild-type yeast, the disruption of BRY1 has no apparent phenotype, but under conditions where the activities of SBF and DSC1/MBF are reduced, BRY1 becomes essential. Our data imply the existence of a third pathway affecting cyclin expression. BRY1 is the same gene as SKN7 which has significant sequence homology to the receiver domains found in response regulator proteins from the bacterial two-component signal transduction pathways. SKN7 is thought to affect cell wall structure, and when highly overexpressed we find that BRY1/SKN7 is lethal perhaps because of perturbations in cell wall biosynthesis. The lethality is partially rescued by genes from the protein kinase C pathway, but genetic data imply that BRY1/SKN7 and protein kinase C are not in the same pathway. Our results suggest that Bry1/Skn7 can influence the expression of MCB- and SCB-driven gene expression in budding yeast, perhaps including genes involved in cell wall metabolism, via a two-component signal transduction pathway which activates Bry1/Skn7 in response to an unidentified signal.

Cyclins↗
PubMed2006

The relative merits of the tetO2 and tetO7 promoter systems for the functional analysis of heterologous genes in yeast and a compilation of essential yeast genes with tetO2 promoter substitutions.

We have generated a collection of yeast strains, each of which has an essential yeast gene under the control of the tetracycline-responsive, tetO, promoter. Screens using first-generation promoter-swap strains uncovered the non-specific responsiveness of the tetO7 promoter to a known human transcription factor (hIRF-1). Non-specific regulation was not observed with the tetO2 promoter. Reporter assays have been used to demonstrate this phenomenon. Subsequent efforts to generate a collection of tetracycline-regulatable strains have focused on the tetO2 promoter. These strains are available to the yeast community and can be used for functional genomics studies.

Bacterial Proteins↗