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At least 217 records · Page 12Linked to original sources

PubMed2005

Cloning and characterization of a dual-specificity kinase gene in rice (Oryza sative).

We identified and isolated a dual-specificity kinase gene,OsSTY kinase (O. sative serine/threonine/tyrosine kinase) gene, from rice. OsSTY kinase gene encoded a protein of 417 amino acids with calculated molecular weight 45926 Da and isoelectric point 7.689. OsSTY kinase is involved in the multiple stresses signaling pathway. Low- and high-temperature (4 degrees C and 37 degrees C) stresses significantly induce the expression of OsSTY kinase. The kinase is also up-regulated upon wounding (by cut), salicylic acid (SA) and ethophon (ET). Moreover,ectopic expression of OsSTY kinase gene in yeast Saccharomyces cerevisiae ste7/ste7 mutant partially suppressed the pseudohyphal development defect of the strain under the condition of nitrogen starvation. Ste7 is a serine/threonine/tyrosine kinase of Saccharomyces cerevisiae, which shares 32% identity and 50% similarity with OsSTY kinase in the kinase domains. This finding suggested that OsSTY kinase could correct, at least partially, the defect of ste7/ste7 mutant.

Amino Acid Sequence↗

PubMed1985

[Effect of cultivation temperature on yeasts under unlimited growth conditions].

The growth specific rate, cells dimensions, the number of cells gemmation, juvenile and postjuvenile development phases duration have been measured on the yeast turbidostate culture Saccharomyces cerevisiae strain 14 growing under different temperature conditions. 33 degrees C has been found to be the optimum temperature. Cells surface area is minimum at that temperature. The number of cells gemmation is the same at suboptimum and optimum temperature but it decreases at supraoptimum one. It has been concluded that the culture growing at 33 degrees C is in the least steady state with dependence typical of the system enduring phase transition.

Culture Media↗

PubMed2006

High-resolution mutation mapping reveals parallel experimental evolution in yeast.

Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2-24 kb (0.1-10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose-galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene.

Algorithms↗

PubMed2025

Evolution and applications of genome-scale metabolic models in yeast systems biology studies.

Genome-scale metabolic models (GEMs) can be used to simulate the metabolic network of an organism in a systematic and holistic way. Different yeast species, including Saccharomyces cerevisiae, have emerged as powerful cell factories for bioproduction. Recently, with the dedicated efforts from the scientific community, significant progress has been made in the development of yeast GEMs. Numerous versions of yeast GEMs and the derived multiscale models have been released, facilitating integrative omics analysis and rational strain design for different types of yeast cell factories. These advancements reflected the evolution and maturation of yeast GEMs together with a model ecosystem around them. This review will summarize the development and expansion of yeast GEMs and discuss their applications in yeast systems biology studies. It is anticipated that yeast GEMs will continue to play an increasingly important role in pioneering yeast physiological and metabolic studies in coming years.

Systems Biology↗

PubMed1977

Yeast protoplasts from stationary and starved cells: preparation, ultrastructure and vacuolar development.

The conversion of stationary and starved yeast cells into protoplasts is described. The method is rapid, simple and can be applied to a variety of stationary yeast cells. Preincubation of yeast cells in the presence of pronase was essential for effective conversion into protoplasts. Baker's yeast and seven defined yeast strains, including one "petite", were studied. All of them were efficiently transformed into protoplasts in 60 to 90 min, depending on the strain culture conditions and the age of the culture. Protoplasts may be obtained even from late-stationary cells which contain spores. Saccharomyces cerevisiae cells subjected to complete starvation conditions in water, could also be completely transformed into protoplasts, even after 48 h of starvation. Electron microscope examination of stationary protoplasts from three different yeast strains showed no evidence of a remaining cell-wall. S. cerevisiae stationary cells show a very developed vacuolar system, a number of "lipid granules" and a few altered mitochondria. Endomycopsis fibuligera and Candida tropicalis stationary protoplasts show a similar fine structure, but "lipid granules" were completely absent.

Culture Media↗

PubMed1996

A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast.

Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation. Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants. Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis. Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP. However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability. Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation. We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP. This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development.

Arabidopsis↗

PubMed2005

Proteomic analysis of ubiquitin conjugates in yeast.

Although the list of proteins modified by ubiquitination has been growing rapidly, a reliable method to biochemically isolate and identify in vivo ubiquitinated substrates is needed. Here we describe a proteomic approach to enrich, identify, and validate ubiquitinated proteins from Saccharomyces cerevisiae on a large scale. To facilitate the purification of ubiquitinated proteins, all four ubiquitin genes were knocked out, and a plasmid coding N-terminal His-tagged ubiquitin was introduced in a yeast strain. Ubiquitinated proteins from the strain were purified by metal chelation chromatography under denaturing condition to minimize co-isolation of interacting proteins. Purified proteins were further analyzed by highly sensitive mass spectrometry to determine their identities. The ubiquitination sites in the proteins could be determined in many cases according to the mass shift caused by the modification. Moreover, the polyubiquitin chain topology could be detected by the same method as well. To confirm the genuineness of the identified ubiquitin conjugates, several strategies were developed: (1) to subtract proteins detected in the control experiment (metal chelation purification from wild-type yeast strain); (2) to remove proteins that did not show the increase of apparent molecular weight because of ubiquitination; (3) to accept proteins with identified ubiquitination sites; and (4) to confirm the state of ubiquitination of individual protein by immunoprecipitation and Western blotting analysis. The method provides a generic approach for biochemical characterization of ubiquitinated proteins and can be extended to analyze targets of ubiquitin-like molecules.

Amino Acid Sequence↗

PubMed2006

Extension of chronological life span in yeast by decreased TOR pathway signaling.

Chronological life span (CLS) in Saccharomyces cerevisiae, defined as the time cells in a stationary phase culture remain viable, has been proposed as a model for the aging of post-mitotic tissues in mammals. We developed a high-throughput assay to determine CLS for approximately 4800 single-gene deletion strains of yeast, and identified long-lived strains carrying mutations in the conserved TOR pathway. TOR signaling regulates multiple cellular processes in response to nutrients, especially amino acids, raising the possibility that decreased TOR signaling mediates life span extension by calorie restriction. In support of this possibility, removal of either asparagine or glutamate from the media significantly increased stationary phase survival. Pharmacological inhibition of TOR signaling by methionine sulfoximine or rapamycin also increased CLS. Decreased TOR activity also promoted increased accumulation of storage carbohydrates and enhanced stress resistance and nuclear relocalization of the stress-related transcription factor Msn2. We propose that up-regulation of a highly conserved response to starvation-induced stress is important for life span extension by decreased TOR signaling in yeast and higher eukaryotes.

Amino Acids↗

PubMed2003

Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae).

Culture medium affected the virulence of a strain of Candida albicans toward Galleria mellonella larvae, but the yeast growth rates in yeast extract - peptone - dextrose broth and synthetic Galleria serum were not correlated with yeast virulence. Virulent C. albicans grew rapidly in larval serum, whereas, it limited nodulation and continued development in vivo, producing toxins that damaged the hemocytes and fat body. Nonpathogenic yeast-phase cells grew slowly in larval serum but induced extensively melanized nodules in vivo and developed no further. There was no discernible relationship in 14 exo-enzymes between the virulent and avirulent yeast strains and virulence. The avirulent myosin-I-defective yeast cells were rapidly removed from the hemolymph in vivo because of lysozyme-mediated yeast agglutination and the possible binding of the yeast cells by lysozyme and apolipophorin-III. Both lysozyme and apolipophorin-III are proteins that bind beta-1,3-glucan. Finally, insects with nonpathogenic C. albicans exhibited induced immunity and were more resistant to candidiasis from the wild-type yeast cells than were noninduced insects.

Animals↗

PubMed1978

Production and ecological significance of yeast cell wall-degrading enzymes from oerskovia.

Motile actinomycetes capable of degrading walls of viable yeast cells were isolated from soil and identified as Oerskovia xanthineolytica. A lytic assay based on susceptibility of enzyme-treated cells to osmotic shock was developed, and 10 of 15 strains of O. xanthineolytica, Oerskovia turbata, and nonmotile Oerskovia- like organisms from other collections were found to possess yeast lytic activities. All lytic strains produced laminaranase and alpha-mannanase, but the amounts, determined by reducing group assays, were not proportional to the observed lytic activities. The Oerskovia isolates demonstrated chemotactic, predatory activity against various yeast strains and killed yeasts in mixed cultures. Of 15 carbon sources tested for production of lytic enzyme, purified yeast cell walls elicited the highest activity. Glucose repressed enzyme production and caused cells to remain in the microfilamentous and motile rod stages of the Oerskovia cell cycle. Crude lytic activity was optimal at pH 5.6 to 7.0 and inactivated by heating for 6 min at 50 degrees C. Partial purification by isoelectric focusing showed that all lytic activity was associated with four beta-(1-->3)-glucanases. The absence of protein disulfide reductase, N-acetyl-beta-d-hexosaminidase, and phosphomannanase in crude preparations indicated that the principal enzyme responsible for yeast wall lysis was a beta-(1-->3)-glucanase that produced relatively little reducing sugar from yeast glucan.

Journal Article↗

PubMed2006

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

DNA Primers↗

PubMed2003

Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants.

A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E,E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E,E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl(2) provided optimum conditions for the stabilization of FPP.

Alkaline Phosphatase↗

PubMed1999

Mass mating method in combination with G418- and aureobasidin A-resistance markers for efficient selection of hybrids from homothallic strains in Saccharomyces cerevisiae.

We have developed a mass mating method using the spore suspensions of homothallic yeasts of Saccharomyces cerevisiae in combination with dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A for the selection of the hybrids. To examine the effectiveness of these markers in the mass mating method, each marker was introduced into a homothallic wine yeast. Using a mixed culture of spore suspensions from the resultant transformants, many hybrids were screened by the drug resistance markers. This method is more practical than the spore-to-spore mating method because it does not require the use of a micromanipulator and many hybrids are obtained at one time. The resultant hybrids could be utilized for industrial brewing because plasmids, which are used to confer resistance markers, are easily eliminated from the hybrids by cultivation in a medium without drugs. We propose that the mass mating method using spore suspensions in combination with dominant selective geneticin- and aureobasidin A-resistance markers is useful for the selection of hybrids from industrial homothallic yeasts.

Journal Article↗

PubMed1988

A synthetic medium for Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae strain Castelli 20 has been observed in a minimal synthetic medium using different glucose concentrations and without biotin, during the first 30 hrs of its development at 28 degrees C. The yeast's growth was observed spectrophotometrically at 675 nm reading the Optical Density every hour. The minimal medium of Sheperd et al. (1980), with glucose 15 g/L and biotin was modified: the vitamin was eliminated and the concentration of glucose was gradually reduced down to 0.5 g/L. At 5 g/L of glucose concentration and without vitamins the strain grew very well. From our results up to 30 hrs biotin has no influence on the yeast's growth. The medium would be useful to study Saccharomyces cerevisiae physiology during the early period of its development.

Biotin↗

PubMed1993

Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae.

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

Alleles↗

PubMed1997

Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism.

The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.

Cyclic AMP↗

PubMed2003

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing.

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.

Bread↗

PubMed1988

[Clinico-experimental study of a dried selective medium for isolating fungi in the genus Candida].

The trial of a newly developed dried selective medium for the isolation of fungi of the genus Candida, involving the inoculation of 103 museum yeast strains and 542 specimens of pathological material, has been carried out. The data obtained in this trial indicate that the proposed medium has advantages over wort agar and Sabouraud medium with antibiotics in the germination index, in the ability to ensure fungal growth after the inoculation of pathological material, and in selective properties. The medium ensures the intactness of the morpho-cultural, biochemical and serological properties of the fungi. The results of the trial recommend the newly developed preparation for laboratory practice for the isolation of yeast-like fungi from clinical material in the diagnosis of candidiasis, intestinal microflora disturbances, as well as for isolation of the fungi from various environmental objects.

Agar↗