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Sample records for promoters saccharomyces cerevisiae

  1. Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

  2. Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

    PubMed

    Rich, Matthew S; Payen, Celia; Rubin, Alan F; Ong, Giang T; Sanchez, Monica R; Yachie, Nozomu; Dunham, Maitreya J; Fields, Stanley

    2016-05-01

    In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach. PMID:26936925

  3. Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis.

    PubMed

    Lee, K Michael; DaSilva, Nancy A

    2005-04-30

    The Saccharomyces cerevisiae ADH2 promoter (P(ADH2)) is repressed several hundred-fold in the presence of glucose; transcription is initiated once the glucose in the medium is exhausted. The promoter can thus be utilized for effective regulation of recombinant gene expression in S. cerevisiae without the addition of an inducer. To evaluate this promoter in the absence of plasmid copy number and stability variations, the P(ADH2)-lacZ cassette was integrated into the yeast chromosomes. The effects of medium composition, glucose concentration and cultivation time on promoter derepression and expression level were investigated. Maximum protein activity was obtained after 48 h of growth in complex YPD medium containing 1% glucose. The widely used S. cerevisiae GAL1 and CUP1 promoters both require the addition of an inducer [galactose and copper(II) ion, respectively] before regulated genes will be expressed. The strengths of these three different promoters were compared for cells containing one copy of an integrated lacZ gene under their control. The ADH2 promoter was superior for all induction strategies investigated. PMID:15849781

  4. Regulation of the Hansenula polymorpha maltase gene promoter in H. polymorpha and Saccharomyces cerevisiae1.

    PubMed

    Alamäe, Tiina; Pärn, Pille; Viigand, Katrin; Karp, Helen

    2003-11-01

    Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Delta failed to grow on maltose and sucrose, showing that maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Delta from the truncated variants of the promoter enabled us to define the 5'-upstream region as sufficient for the induction of maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Delta from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter. PMID:14613881

  5. Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene.

    PubMed

    Joo, Yoo Jin; Kim, Jin-Ha; Baek, Joung Hee; Seong, Ki Moon; Lee, Jae Yung; Kim, Joon

    2009-01-01

    Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using beta-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene. PMID:19853675

  6. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae.

    PubMed

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen; Sommer, Morten O A

    2015-12-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity. Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae. Utilizing 3vGFP, we further engineered a less leaky Cu(2+)-inducible promoter based on CUP1. The basal expression level of the new promoter was approximately 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu(2+)-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms. PMID:26392044

  7. AFR1 promotes polarized apical morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Konopka, J B; DeMattei, C; Davis, C

    1995-01-01

    The G protein-coupled alpha-factor receptor promotes polarized growth toward a mating partner. alpha-Factor induces the expression of AFR1, which acts together with the receptor C terminus to promote normal morphogenesis. The function of AFR1 was investigated by engineering cells to constitutively express AFR1 without alpha-factor. Constitutive AFR1 expression caused cells to form elongated buds that demonstrate that AFR1 can also interact with the morphogenesis components that promote bud formation. A similar elongated bud phenotype is caused by mutation of the CDC3, CDC10, CDC11, and CDC12 genes, which encode putative filament proteins that form a ring at the bud neck. AFR1 may act directly on the filament proteins, since immunolocalization detected AFR1 at the bud neck and interaction of AFR1 and CDC12 was detected in the two-hybrid protein assay. AFR1 localized to the base of pheromone-induced projections. These results suggest that AFR1 and the putative filament proteins act together with the receptor to facilitate proper localization of components during mating. PMID:7823940

  8. Bacterial XylRs and synthetic promoters function as genetically encoded xylose biosensors in Saccharomyces cerevisiae.

    PubMed

    Teo, Wei Suong; Chang, Matthew Wook

    2015-02-01

    Lignocellulosic biomass is a sustainable and abundant starting material for biofuel production. However, lignocellulosic hydrolysates contain not only glucose, but also other sugars including xylose which cannot be metabolized by the industrial workhorse Saccharomyces cerevisiae. Hence, engineering of xylose assimilating S. cerevisiae has been much studied, including strain optimization strategies. In this work, we constructed genetically encoded xylose biosensors that can control protein expression upon detection of xylose sugars. These were constructed with the constitutive expression of heterologous XylR repressors, which function as protein sensors, and cloning of synthetic promoters with XylR operator sites. Three XylR variants and the corresponding synthetic promoters were used: XylR from Tetragenococcus halophile, Clostridium difficile, and Lactobacillus pentosus. To optimize the biosensor, two promoters with different strengths were used to express the XylR proteins. The ability of XylR to repress yEGFP expression from the synthetic promoters was demonstrated. Furthermore, xylose sugars added exogenously to the cells were shown to regulate gene expression. We envision that the xylose biosensors can be used as a tool to engineer and optimize yeast that efficiently utilizes xylose as carbon source for growth and biofuel production. PMID:24975936

  9. Heat Shock Protein Genes and Newly Integrated Glucose Metabolic Pathways Promote Ethanol Tolerance of Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulose-to-ethanol conversion provides a promising alternative means for production of sustainable and cleaner transportation fuels. Development of stress tolerant ethanologenic Saccharomyces cerevisiae is important for low-cost biobased economy. Tolerance to high levels of ethanol has been...

  10. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. PMID:27184763

  11. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity. PMID:25142154

  12. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  13. Specific interactions of Saccharomyces cerevisiae proteins with a promoter region of eukaryotic tRNA genes.

    PubMed Central

    Klemenz, R; Stillman, D J; Geiduschek, E P

    1982-01-01

    The specific binding of one or several Saccharomyces cerevisiae proteins to a segment of genes that code for different yeast tRNAs has been demonstrated with the use of the DNase I-protection "footprint" assay of Galas and Schmitz. The analyzed binding occurs near the 3' ends of the genes and is centered on an 11-base-pair DNA sequence that has been well conserved among eukaryotic tRNA genes. Others have shown the involvement of this sequence in initiating the transcription of tRNA genes by RNA polymerase III. The adenovirus gene that codes for VAI RNA also contains this conserved sequence element, and we detect binding of yeast protein(s) to this gene. Competition experiments show that a common set of proteins binds to different tRNA genes. The DNA-protein complex is quite stable at 20 degrees C and low ionic strength. Images PMID:6755466

  14. Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria

    PubMed Central

    Roloff, Gabrielle A.; Henry, Michael F.

    2015-01-01

    Three mitochondrial DNA–encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. PMID:26108620

  15. Preparation of Saccharomyces cerevisiae expression plasmids.

    PubMed

    Drew, David; Kim, Hyun

    2012-01-01

    Expression plasmids for Saccharomyces cerevisiae offer a wide choice of vector copy number, promoters of varying strength and selection markers. These expression plasmids are usually shuttle vectors that can be propagated both in yeast and bacteria, making them useful in gene cloning. For heterologous production of membrane proteins, we used the green fluorescent protein (GFP) fusion technology which was previously developed in the Escherichia coli system. We designed an expression plasmid carrying an inducible GAL1 promoter, a gene encoding a membrane protein of interest and the GFP-octa-histidine sequence. Here we describe construction of multi-copy yeast expression plasmids by homologous recombination in S. cerevisiae. PMID:22454112

  16. Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.

    PubMed

    Kim, Sujin; Lee, Kyusung; Bae, Sang-Jeong; Hahn, Ji-Sook

    2015-03-01

    A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering. PMID:25573467

  17. Regulatory regions in the promoters of the Saccharomyces cerevisiae PYC1 and PYC2 genes encoding isoenzymes of pyruvate carboxylase.

    PubMed

    Menéndez, J; Gancedo, C

    1998-07-15

    We have identified regions in the promoters of the PYC1 and PYC2 genes from Saccharomyces cerevisiae involved in their regulation in different culture conditions. In the case of PYC1, a UAS in the region between -330/-297 and three repressing sequences with the common central core CCGCC at positions -457, -432 and -399 were identified. Specific binding of nuclear proteins to the -330/-214 DNA fragment was abolished in rtg mutants suggesting a role for the RTG genes in the control of PYC1 expression. In the case of the PYC2 promoter, elimination of a fragment from -417 to -291 brings about a two-fold decrease in the expression in repressed conditions and a similar increase in derepression. PMID:9682484

  18. Identification of a 450-bp region of human papillomavirus type 1 that promotes episomal replication in Saccharomyces cerevisiae

    SciTech Connect

    Chattopadhyay, Anasuya; Schmidt, Martin C.; Khan, Saleem A. . E-mail: khan@pitt.edu

    2005-09-15

    Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3' end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783-7232) that includes 293 nucleotides from the 5' region of the viral LCR and 157 nucleotides from the 3' end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae.

  19. Peptidase activities in Saccharomyces cerevisiae.

    PubMed Central

    Rose, B; Becker, J M; Naider, F

    1979-01-01

    At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives. Images PMID:378955

  20. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

    Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production. PMID:10704473

  1. Cadmium biosorption by Saccharomyces cerevisiae

    SciTech Connect

    Volesky, B.; May, H.; Holan, Z.R. )

    1993-04-01

    Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium-bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal-sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min.

  2. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    PubMed

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. PMID:27188886

  3. Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae.

    PubMed

    Mutlu, Nebibe; Garipler, Görkem; Akdoğan, Emel; Dunn, Cory D

    2014-07-01

    Genetic and microscopic approaches using Saccharomyces cerevisiae have identified many proteins that play a role in mitochondrial dynamics, but it is possible that other proteins and pathways that play a role in mitochondrial division and fusion remain to be discovered. Mutants lacking mitochondrial fusion are characterized by rapid loss of mitochondrial DNA. We took advantage of a petite-negative mutant that is unable to survive mitochondrial DNA loss to select for mutations that allow cells with fusion-deficient mitochondria to maintain the mitochondrial genome on fermentable medium. Next-generation sequencing revealed that all identified suppressor mutations not associated with known mitochondrial division components were localized to PDR1 or PDR3, which encode transcription factors promoting drug resistance. Further studies revealed that at least one, if not all, of these suppressor mutations dominantly increases resistance to known substrates of the pleiotropic drug resistance pathway. Interestingly, hyperactivation of this pathway did not significantly affect mitochondrial shape, suggesting that mitochondrial division was not greatly affected. Our results reveal an intriguing genetic connection between pleiotropic drug resistance and mitochondrial dynamics. PMID:24807265

  4. The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

    PubMed Central

    2011-01-01

    Background The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation. PMID:22204397

  5. Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter.

    PubMed

    Bahieldin, Ahmed; Gadalla, Nour O; Al-Garni, Saleh M; Almehdar, Hussein; Noor, Samah; Hassan, Sabah M; Shokry, Ahmed M; Sabir, Jamal S M; Murata, Norio

    2014-03-01

    Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate. PMID:24680933

  6. PET genes of Saccharomyces cerevisiae.

    PubMed Central

    Tzagoloff, A; Dieckmann, C L

    1990-01-01

    We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae. PMID:2215420

  7. Methionine catabolism in Saccharomyces cerevisiae.

    PubMed

    Perpète, Philippe; Duthoit, Olivier; De Maeyer, Simon; Imray, Louise; Lawton, Andrew I; Stavropoulos, Konstantinos E; Gitonga, Virginia W; Hewlins, Michael J E; Dickinson, J Richard

    2006-01-01

    The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol. PMID:16423070

  8. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  9. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  10. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

    Resnick, M.A.; Boyce, J.; Cox, B.

    1981-04-01

    Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light. Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were chased, the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, it was concluded that postreplication repair in excision-defective mutants does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.

  11. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

    Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua

    2002-01-01

    Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

  12. Saccharomyces cerevisiae osteomyelitis in an immunocompetent baker.

    PubMed

    Seng, Piseth; Cerlier, Alexandre; Cassagne, Carole; Coulange, Mathieu; Legré, Regis; Stein, Andreas

    2016-01-01

    Invasive infection caused by Saccharomyces cerevisiae is rare. We report the first case of osteomyelitis caused by S. cerevisiae (baker's yeast) in a post-traumatic patient. The clinical outcome was favorable after surgical debridement, prolonged antifungal treatment and hyperbaric oxygen therapy. PMID:27347482

  13. Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.

    PubMed

    Leavitt, John M; Tong, Alice; Tong, Joyce; Pattie, Jonathan; Alper, Hal S

    2016-07-01

    Gene expression requires the coordination of trans-acting factors and cis-DNA elements to initiate transcription. Here we present a coordinated approach that combines cis-acting element engineering with mutant trans-acting factors to engineer yeast promoters. Specifically, we first construct a hybrid promoter based on the ARO9 upstream region that exhibits high constitutive and inducible expression with respect to exogenous tryptophan. Next, we perform protein engineering to identify a mutant Aro80p that affords both high constitutive expression while retaining inducible traits. We then use this mutant trans-acting factor to drive expression and generate ultra-strong promoters with transcriptional output roughly 2 fold higher than TDH3 (GPD), one of the strongest promoters to-date. Finally, we used this element to construct a modular expression system capable of staged outputs resulting in a system with nearly 6-fold, 12-fold and 15-fold expression relative to the off-state. This work further highlights the potential of using endogenous transcription factors (including mutant factors) along with hybrid promoters to expand the yeast synthetic biology toolbox. PMID:27152757

  14. Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae.

    PubMed Central

    Kim, S; Mellor, J; Kingsman, A J; Kingsman, S M

    1986-01-01

    The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II. The difference in size between the largest and smallest mRNAs is about 200 base pairs. A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting. We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains. The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements. The negative element, mapped between -293 and -318, expression of transcript I. Two regions of transcript I. Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I. Each region contained sequences homologous to known consensus sequences of the TATA box. Images PMID:3025648

  15. Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Hong, Soogil; Kim, Keun Pil

    2013-01-01

    Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias. PMID:24213600

  16. Saccharomyces cerevisiae MPT5 and SSD1 function in parallel pathways to promote cell wall integrity.

    PubMed Central

    Kaeberlein, Matt; Guarente, Leonard

    2002-01-01

    Yeast MPT5 (UTH4) is a limiting component for longevity. We show here that MPT5 also functions to promote cell wall integrity. Loss of Mpt5p results in phenotypes associated with a weakened cell wall, including sorbitol-remedial temperature sensitivity and sensitivities to calcofluor white and sodium dodecyl sulfate. Additionally, we find that mutation of MPT5, in the absence of SSD1-V, is lethal in combination with loss of either Ccr4p or Swi4p. These synthetic lethal interactions are suppressed by the SSD1-V allele. Furthermore, we have provided evidence that the short life span caused by loss of Mpt5p is due to a weakened cell wall. This cell wall defect may be the result of abnormal chitin biosynthesis or accumulation. These analyses have defined three genetic pathways that function in parallel to promote cell integrity: an Mpt5p-containing pathway, an Ssd1p-containing pathway, and a Pkc1p-dependent pathway. This work also provides evidence that post-transcriptional regulation is likely to be important both for maintaining cell integrity and for promoting longevity. PMID:11805047

  17. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  18. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  19. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  20. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  1. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  2. Conversion of starch to ethanol in a recombinant saccharomyces cerevisiae strain expressing rice [alpha]-amylase from a novel Pichia pastoris alcohol oxidase promoter

    SciTech Connect

    Kumagai, M.H.; Sverlow, G.G.; della-Cioppa, G.; Grill, L.K. )

    1993-05-01

    A recombinant Saccharomyces cerevisiae, expressing and secreting rice [alpha]-amylase, converts starch to ethanol. The rice [alpha]-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N[sub 3]-GCTTCCAA-N[sub 5]-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida biodinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch. 45 refs., 8 figs.

  3. Asymmetrical division of Saccharomyces cerevisiae.

    PubMed Central

    Lord, P G; Wheals, A E

    1980-01-01

    The unequal division model proposed for budding yeast (L. H. Hartwell and M. W. Unger, J. Cell Biol. 75:422-435, 1977) was tested by bud scar analyses of steady-state exponential batch cultures of Saccharomyces cerevisiae growing at 30 degrees C at 19 different rates, which were obtained by altering the carbon source. The analyses involved counting the number of bud scars, determining the presence or absence of buds on at least 1,000 cells, and independently measuring the doubling times (gamma) by cell number increase. A number of assumptions in the model were tested and found to be in good agreement with the model. Maximum likelihood estimates of daughter cycle time (D), parent cycle time (P), and the budded phase (B) were obtained, and we concluded that asymmetrical division occurred at all growth rates tested (gamma, 75 to 250 min). D, P, and B are all linearly related to gamma, and D, P, and gamma converge to equality (symmetrical division) at gamma = 65 min. Expressions for the genealogical age distribution for asymmetrically dividing yeast cells were derived. The fraction of daughter cells in steady-state populations is e-alpha P, and the fraction of parent cells of age n (where n is the number of buds that a cell has produced) is (e-alpha P)n-1(1-e-alpha P)2, where alpha = IN2/gamma; thus, the distribution changes with growth rate. The frequency of cells with different numbers of bud scars (i.e., different genealogical ages) was determined for all growth rates, and the observed distribution changed with the growth rate in the manner predicted. In this haploid strain new buds formed adjacent to the previous buds in a regular pattern, but at slower growth rates the pattern was more irregular. The median volume of the cells and the volume at start in the cell cycle both increased at faster growth rates. The implications of these findings for the control of the cell cycle are discussed. PMID:6991494

  4. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant eff...

  5. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

    Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.

    2008-01-01

    Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…

  6. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

    PubMed Central

    Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B

    1992-01-01

    The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity. Images PMID:1735719

  7. The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

    PubMed

    Ye, Yanfang; Kirkham-McCarthy, Lucy; Lahue, Robert S

    2016-07-01

    Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11. PMID:27173583

  8. The intrinsic topological information of the wild-type and of up-promoter mutations of the Saccharomyces cerevisiae alcohol dehydrogenase II regulatory region.

    PubMed

    Della Seta, F; Camilloni, G; Venditti, S; Di Mauro, E

    1988-11-01

    A 569-base pair fragment encompassing the upstream regulatory region, the RNA initiation sites, and the initial part of the coding region of the Saccharomyces cerevisiae alcohol dehydrogenase II gene has been analyzed for the presence of sites which undergo conformational modification under torsional stress. Fine mapping of P1 and S1 endonuclease-sensitive sites was obtained on single topoisomers produced by in vitro ligation. It was shown that the upstream activator sequence, the TATA sequence, a region directly upstream to the RNA initiation sites, and several positions in the first segment of the transcribed region change conformation as a function of the applied torsional stress in a precisely coordinate fashion. The superhelical density optima for this coordinate modifications have been determined. Analysis of the conformational changes of the promoter sequence in several naturally occurring (Young, E. T., Williamson, V. M., Taguchi, A., Smith, M., Sledziewski, L., Russel, D., Osterman, J., Denis, C., Cox, D., and Beier, D., (1982) in Genetic Engineering of Microorganisms for Chemicals (Hollander, A., De Moss, R. D., Kaplan, S., Konisky, J., Savage, D., and Wolle, R. S., eds) pp. 335-361, Plenum Publishing Corp., New York) up-promoter constitutive mutants was performed. This analysis has shown that the conformation of functionally relevant sites changes as a function of sequence mutations that have taken place elsewhere; this shows that the conformational behavior of the whole promoter region is linked and suggests transmission in cis of topological effects in RNA polymerase II promoters. PMID:3053683

  9. Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Bae, Ju Yun; Laplaza, José; Jeffries, Thomas W.

    Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled d-xylose reductase (XYL1) and d-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (l→2→ or √1√2), convergent (1→√2), and divergent (√1 2→) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then coexpressed XYL2 from either a single or multicopy plasmid, which was also driven by the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYLl was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

  10. [Urinary infection by Saccharomyces cerevisiae: Emerging yeast?].

    PubMed

    Elkhihal, B; Elhalimi, M; Ghfir, B; Mostachi, A; Lyagoubi, M; Aoufi, S

    2015-12-01

    Saccharomyces cerevisiae is a commensal yeast of the digestive, respiratory and genito-urinary tract. It is widely used as a probiotic for the treatment of post-antibiotic diarrhea. It most often occurs in immunocompromised patients frequently causing fungemia. We report the case of an adult diabetic patient who had a urinary tract infection due to S. cerevisiae. The disease started with urination associated with urinary frequency burns without fever. The diagnosis was established by the presence of yeasts on direct examination and positivity of culture on Sabouraud-chloramphenicol three times. The auxanogramme gallery (Auxacolor BioRad(®)) allowed the identification of S. cerevisiae. The patient was put on fluconazole with good outcome. This observation points out that this is an opportunistic yeast in immunocompromised patients. PMID:26522963

  11. Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.

    PubMed

    Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung

    2015-08-01

    Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). PMID:26220934

  12. Specific components of the SAGA complex are required for Gcn4- and Gcr1-mediated activation of the his4-912delta promoter in Saccharomyces cerevisiae.

    PubMed Central

    Dudley, A M; Gansheroff, L J; Winston, F

    1999-01-01

    Mutations selected as suppressors of Ty or solo delta insertion mutations in Saccharomyces cerevisiae have identified several genes, SPT3, SPT7, SPT8, and SPT20, that encode components of the SAGA complex. However, the mechanism by which SAGA activates transcription of specific RNA polymerase II-dependent genes is unknown. We have conducted a fine-structure mutagenesis of one widely used SAGA-dependent promoter, the delta element of his4-912delta, to identify sequence elements important for its promoter activity. Our analysis has characterized three delta regions necessary for full promoter activity and accurate start site selection: an upstream activating sequence, a TATA region, and an initiator region. In addition, we have shown that factors present at the adjacent UASHIS4 (Gcn4, Bas1, and Pho2) also activate the delta promoter in his4-912delta. Our results suggest a model in which the delta promoter in his4-912delta is primarily activated by two factors: Gcr1 acting at the UASdelta and Gcn4 acting at the UASHIS4. Finally, we tested whether activation by either of these factors is dependent on components of the SAGA complex. Our results demonstrate that Spt3 and Spt20 are required for full delta promoter activity, but that Gcn5, another member of SAGA, is not required. Spt3 appears to be partially required for activation of his4-912delta by both Gcr1 and Gcn4. Thus, our work suggests that SAGA exerts a large effect on delta promoter activity through a combination of smaller effects on multiple factors. PMID:10101163

  13. Sporulation in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Neiman, Aaron M.

    2011-01-01

    In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423

  14. Association of Transcription Factor IIA with TATA Binding Protein Is Required for Transcriptional Activation of a Subset of Promoters and Cell Cycle Progression in Saccharomyces cerevisiae

    PubMed Central

    Ozer, Josef; Lezina, Larissa E.; Ewing, Joshua; Audi, Salma; Lieberman, Paul M.

    1998-01-01

    The general transcription factor IIA (TFIIA) interacts with the TATA binding protein (TBP) and promoter DNA to mediate transcription activation in vitro. To determine if this interaction is generally required for activation of all class II genes in vivo, we have constructed substitution mutations in yeast TFIIA which compromise its ability to bind TBP. Substitution mutations in the small subunit of TFIIA (Toa2) at residue Y69 or W76 significantly impaired the ability of TFIIA to stimulate TBP-promoter binding in vitro. Gene replacement of wild-type TOA2 with a W76E or Y69A/W76A mutant was lethal in Saccharomyces cerevisiae, while the Y69F/W76F mutant exhibited extremely slow growth at 30°C. Both the Y69A and W76A mutants were conditionally lethal at higher temperatures. Light microscopy indicated that viable toa2 mutant strains accumulate as equal-size dumbbells and multibudded clumps. Transcription of the cell cycle-regulatory genes CLB1, CLB2, CLN1, and CTS1 was significantly reduced in the toa2 mutant strains, while the noncycling genes PMA1 and ENO2 were only modestly affected, suggesting that these toa2 mutant alleles disrupt cell cycle progression. The differential effect of these toa2 mutants on gene transcription was examined for a number of other genes. toa2 mutant strains supported high levels of CUP1, PHO5, TRP3, and GAL1 gene activation, but the constitutive expression of DED1 was significantly reduced. Activator-induced start site expression for HIS3, GAL80, URA1, and URA3 promoters was defective in toa2 mutant strains, suggesting that the TFIIA-TBP complex is important for promoters which require an activator-dependent start site selection from constitutive to regulated expression. We present evidence to indicate that transcription defects in toa2 mutants can be both activator and promoter dependent. These results suggest that the association of TFIIA with TBP regulates activator-induced start site selection and cell cycle progression in S

  15. Calcium control of Saccharomyces cerevisiae actin assembly.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo. Images PMID:6757718

  16. Biosorption of heavy metals by Saccharomyces cerevisiae.

    PubMed

    Volesky, B; May-Phillips, H A

    1995-01-01

    Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4-5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu approximately (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast (approximately 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine needle-like crystals both on the inside and outside of the S. cerevisiae cells. PMID:7765919

  17. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate. PMID:18772282

  18. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae

    PubMed Central

    Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J. J.

    2015-01-01

    Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes. PMID:25905794

  19. Myo-inositol transport in Saccharomyces cerevisiae.

    PubMed

    Nikawa, J; Nagumo, T; Yamashita, S

    1982-05-01

    myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide. PMID:7040334

  20. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    PubMed

    Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J J

    2015-01-01

    Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes. PMID:25905794

  1. Energy-dependent effects of resveratrol in Saccharomyces cerevisiae.

    PubMed

    Madrigal-Perez, Luis Alberto; Canizal-Garcia, Melina; González-Hernández, Juan Carlos; Reynoso-Camacho, Rosalia; Nava, Gerardo M; Ramos-Gomez, Minerva

    2016-06-01

    The metabolic effects induced by resveratrol have been associated mainly with the consumption of high-calorie diets; however, its effects with standard or low-calorie diets remain unclear. To better understand the interactions between resveratrol and cellular energy levels, we used Saccharomyces cerevisiae as a model. Herein it is shown that resveratrol: (a) decreased cell viability in an energy-dependent manner; (b) lessening of cell viability occurred specifically when cells were under cellular respiration; and (c) inhibition of oxygen consumption in state 4 occurred at low and standard energy levels, whereas at high energy levels oxygen consumption was promoted. These findings indicate that the effects of resveratrol are dependent on the cellular energy status and linked to metabolic respiration. Importantly, our study also revealed that S. cerevisiae is a suitable and useful model to elucidate the molecular targets of resveratrol under different nutritional statuses. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26945517

  2. Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.

    PubMed

    Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T

    2001-10-01

    Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae. PMID:11761363

  3. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle. PMID:26519319

  4. Transcriptional Regulatory Networks in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Lee, Tong Ihn; Rinaldi, Nicola J.; Robert, François; Odom, Duncan T.; Bar-Joseph, Ziv; Gerber, Georg K.; Hannett, Nancy M.; Harbison, Christopher T.; Thompson, Craig M.; Simon, Itamar; Zeitlinger, Julia; Jennings, Ezra G.; Murray, Heather L.; Gordon, D. Benjamin; Ren, Bing; Wyrick, John J.; Tagne, Jean-Bosco; Volkert, Thomas L.; Fraenkel, Ernest; Gifford, David K.; Young, Richard A.

    2002-10-01

    We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.

  5. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

    The killer systems of Saccharomyces cerevisiae are an unusual class of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. They are characterized by the linearity of the genome, its fragmentation into a major and a minor fraction, which replicate separately, and their ability to control the synthesis of secretory mycocin proteins possessing a toxic action on closely related strains. The secretion of mycocins at the same time ensures acquiring of resistance to them. Strains containing killer symbionts are toxigenic and resistant to the action of their own toxin, but strains that are free of killer double-stranded RNAs are sensitive to the action of mycocins. The killer systems of S. cerevisiae have retained features relating them to viruses and are apparently the result of evolution of infectious viruses. The occurrences of such systems among monocellular eukaryotic organisms is an example of complication of the genome by means of its assembly from virus-like components. We discuss the unusual features of replication and the expression of killer systems and their utilization in the construction of vector molecules.

  6. Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

    PubMed

    Shen, Qing-Ji; Kassim, Hakimi; Huang, Yong; Li, Hui; Zhang, Jing; Li, Guang; Wang, Peng-Ye; Yan, Jun; Ye, Fangfu; Liu, Ji-Long

    2016-06-20

    Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated. PMID:27312010

  7. Force Sensitivity in Saccharomyces cerevisiae Flocculins

    PubMed Central

    Chan, Cho X. J.; El-Kirat-Chatel, Sofiane; Joseph, Ivor G.; Jackson, Desmond N.; Ramsook, Caleen B.; Dufrêne, Yves F.

    2016-01-01

    ABSTRACT Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca2+, yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends

  8. Force Sensitivity in Saccharomyces cerevisiae Flocculins.

    PubMed

    Chan, Cho X J; El-Kirat-Chatel, Sofiane; Joseph, Ivor G; Jackson, Desmond N; Ramsook, Caleen B; Dufrêne, Yves F; Lipke, Peter N

    2016-01-01

    Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on

  9. Prediction of Saccharomyces cerevisiae replication origins

    PubMed Central

    Breier, Adam M; Chatterji, Sourav; Cozzarelli, Nicholas R

    2004-01-01

    Background Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. Results We developed an algorithm, Oriscan, to predict yeast origins using similarity to 26 characterized origins. Oriscan used 268 bp of sequence, including the T-rich ACS and a 3' A-rich region. The predictions identified the exact location of the ACS. A total of 84 of the top 100 Oriscan predictions, and 56% of the top 350, matched known ARSs or replication protein binding sites. The true accuracy was even higher because we tested 25 discrepancies, and 15 were in fact ARSs. Thus, 94% of the top 100 predictions and an estimated 70% of the top 350 were correct. We compared the predictions to corresponding sequences in related Saccharomyces species and found that the ACSs of experimentally supported predictions show significant conservation. Conclusions The high accuracy of the predictions indicates that we have defined near-sufficient conditions for ARS activity, the A-rich region is a recognizable feature of ARS elements with a probable role in replication initiation, and nucleotide sequence is a reliable predictor of yeast origins. Oriscan detected most origins in the genome, demonstrating previously unrecognized generality in yeast replication origins and significant discriminatory power in the algorithm. PMID:15059255

  10. The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

    NASA Astrophysics Data System (ADS)

    Yi, Zong-Chun; Li, Xiao-Fei; Wang, Yan; Wang, Jie; Sun, Yan; Zhuang, Feng-Yuan

    2011-06-01

    The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.

  11. The uptake of different iron salts by the yeast Saccharomyces cerevisiae.

    PubMed

    Gaensly, Fernanda; Picheth, Geraldo; Brand, Debora; Bonfim, Tania M B

    2014-01-01

    Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended. PMID:25242932

  12. Construction of ploidy series of Saccharomyces cerevisiae by the plasmid YCplac33-GHK.

    PubMed

    Hou, Lihua; Li, Xiaoyang; Wang, Cong; Cao, Xiaohong; Wang, Haiyong

    2013-04-01

    An effective approach, using the plasmid YCplac33-GHK, is developed to construct a ploidy series of Saccharomyces cerevisiae. YCplac33-GHK harbors the HO gene under the control of galactose-inducible promoter and KanMX4 as the selective marker. The simple method can solve the problem of industrial applications of strains with resistance genes. PMID:23430413

  13. Regulation of Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Waechter, Charles J.; Lester, Robert L.

    1971-01-01

    Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate 14C-CH3-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of 14C-CH3-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium. Images PMID:5547992

  14. Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Culbertson, Michael R.; Henry, Susan A.

    1975-01-01

    Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974). PMID:1093935

  15. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Werner-Washburne, M; Braun, E; Johnston, G C; Singer, R A

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase. PMID:8393130

  16. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... controls). The special control is FDA's “Guidance for Industry and FDA Reviewers: Class II Special...

  17. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae.

    PubMed

    Comitini, Francesca; Gobbi, Mirko; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio

    2011-08-01

    Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation). PMID:21569929

  18. Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

    PubMed Central

    2012-01-01

    Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most

  19. Regulation of Cation Balance in Saccharomyces cerevisiae

    PubMed Central

    Cyert, Martha S.; Philpott, Caroline C.

    2013-01-01

    All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, the divalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that are regulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment. PMID:23463800

  20. Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas

    PubMed Central

    King, Nichole L; Deutsch, Eric W; Ranish, Jeffrey A; Nesvizhskii, Alexey I; Eddes, James S; Mallick, Parag; Eng, Jimmy; Desiere, Frank; Flory, Mark; Martin, Daniel B; Kim, Bong; Lee, Hookeun; Raught, Brian; Aebersold, Ruedi

    2006-01-01

    We present the Saccharomyces cerevisiae PeptideAtlas composed from 47 diverse experiments and 4.9 million tandem mass spectra. The observed peptides align to 61% of Saccharomyces Genome Database (SGD) open reading frames (ORFs), 49% of the uncharacterized SGD ORFs, 54% of S. cerevisiae ORFs with a Gene Ontology annotation of 'molecular function unknown', and 76% of ORFs with Gene names. We highlight the use of this resource for data mining, construction of high quality lists for targeted proteomics, validation of proteins, and software development. PMID:17101051

  1. Invasive Saccharomyces cerevisiae infection: a friend turning foe?

    PubMed

    Pillai, Unnikrishnan; Devasahayam, Joe; Kurup, Aparna Narayana; Lacasse, Alexandre

    2014-11-01

    We report a very rare case of acute pyelonephritis in a 51-year-old female with a history of chronic kidney disease (CKD) and diabetes caused by a normally benign and a well-known human commensal organism, Saccharomyces cerevisiae that is very often prescribed as a probiotic in modern medical practice. The causal role of S. cerevisiae was confirmed by its isolation in blood, urine, stool as well as vaginal swabs thus proving its virulent nature in suitable situations. PMID:25394448

  2. A global topology map of the Saccharomyces cerevisiae membrane proteome

    NASA Astrophysics Data System (ADS)

    Kim, Hyun; Melén, Karin; Österberg, Marie; von Heijne, Gunnar

    2006-07-01

    The yeast Saccharomyces cerevisiae is, arguably, the best understood eukaryotic model organism, yet comparatively little is known about its membrane proteome. Here, we report the cloning and expression of 617 S. cerevisiae membrane proteins as fusions to a C-terminal topology reporter and present experimentally constrained topology models for 546 proteins. By homology, the experimental topology information can be extended to 15,000 membrane proteins from 38 fully sequenced eukaryotic genomes. membrane proteins | membrane proteomics | yeast

  3. Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production.

    PubMed

    Blazeck, John; Miller, Jarrett; Pan, Anny; Gengler, Jon; Holden, Clinton; Jamoussi, Mariam; Alper, Hal S

    2014-10-01

    Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. Here, we utilize Saccharomyces cerevisiae to enable the synthesis of itaconic acid, a molecule with diverse applications as a petrochemical replacement. We first optimize pathway expression within S. cerevisiae through the use of a hybrid promoter. Next, we utilize sequential, in silico computational genome-scanning to identify beneficial genetic perturbations that are metabolically distant from the itaconic acid synthesis pathway. In this manner, we successfully identify three non-obvious genetic targets (∆ade3 ∆bna2 ∆tes1) that successively improve itaconic acid titer. We establish that focused manipulations of upstream pathway enzymes (localized refactoring) and enzyme re-localization to both mitochondria and cytosol fail to improve itaconic acid titers. Finally, we establish a higher cell density fermentation that ultimately achieves itaconic acid titer of 168 mg/L, a sevenfold improvement over initial conditions. This work represents an attempt to increase itaconic acid production in yeast and demonstrates the successful utilization of computationally guided genetic manipulation to increase metabolic capacity. PMID:24997118

  4. Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Smith, S J; Crowley, J H; Parks, L W

    1996-01-01

    Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae. PMID:8816455

  5. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Osterlund, Tobias; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2013-04-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously. PMID:23208612

  6. [Tolerance of Saccharomyces cerevisiae to monoterpenes--a review].

    PubMed

    Liu, Jidong; Zhou, Jingwen; Chen, Jian

    2013-06-01

    Tolerance of Saccharomyces cerevisiae to monoterpenes is important in both metabolic engineering of the yeast to produce these chemicals de novo and efficient use of biomass containing these chemicals. Understanding the mechanisms in the tolerance of S. cerevisiae to monoterpenes could facilitate the construction of yeast strains with enhanced monoterpenes resistance, and therefore improve related bioprocesses. Monoterpenes could disturb the redox balance in S. cerevisiae, therefore increase the accumulation of reactive oxygen species (ROS) and result in cell death. S. cerevisiae has to systematically improve its antioxidative ability to deal with the ROS induced damage. The current review summarized the recent developments in demonstration of the tolerance of S. cerevisiae to different typical monoterpenes mainly from the aspect of the antioxidative mechanisms. Based on the analysis of the previous works, further attempts to demonstrate the mechanisms were proposed. PMID:24028054

  7. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  8. Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.

    PubMed

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2015-08-01

    During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation. PMID:25956738

  9. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  10. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficient utilization of all available sugars in lignocellulosic biomass, which is more abundant than available commodity crops and starch, represents one of the most difficult technological challenges for the production of bioethanol. The well-studied yeast Saccharomyces cerevisiae has played a...

  11. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yeast Saccharomyces cerevisiae is a superb ethanol producer, yet sensitive to ethanol at higher concentrations especially under high gravity or very high gravity fermentation conditions. Although significant efforts have been made to study ethanol-stress response in past decades, molecular mecha...

  12. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media. PMID:18245248

  13. Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae.

    PubMed

    Petty, Emily L; Lafon, Anne; Tomlinson, Shannon L; Mendelsohn, Bryce A; Pillus, Lorraine

    2016-08-01

    Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability. PMID:27317677

  14. The zinc cluster proteins Upc2 and Ecm22 promote filamentation in Saccharomyces cerevisiae by sterol biosynthesis-dependent and -independent pathways.

    PubMed

    Woods, Kelly; Höfken, Thomas

    2016-02-01

    The transition between a unicellular yeast form to multicellular filaments is crucial for budding yeast foraging and the pathogenesis of many fungal pathogens such as Candida albicans. Here, we examine the role of the related transcription factors Ecm22 and Upc2 in Saccharomyces cerevisiae filamentation. Overexpression of either ECM22 or UPC2 leads to increased filamentation, whereas cells lacking both ECM22 and UPC2 do not exhibit filamentous growth. Ecm22 and Upc2 positively control the expression of FHN1, NPR1, PRR2 and sterol biosynthesis genes. These genes all play a positive role in filamentous growth, and their expression is upregulated during filamentation in an Ecm22/Upc2-dependent manner. Furthermore, ergosterol content increases during filamentous growth. UPC2 expression also increases during filamentation and is inhibited by the transcription factors Sut1 and Sut2. The expression of SUT1 and SUT2 in turn is under negative control of the transcription factor Ste12. We suggest that during filamentation Ste12 becomes activated and reduces SUT1/SUT2 expression levels. This would result in increased UPC2 levels and as a consequence to transcriptional activation of FHN1, NPR1, PRR2 and sterol biosynthesis genes. Higher ergosterol levels in combination with the proteins Fhn1, Npr1 and Prr2 would then mediate the transition to filamentous growth. PMID:26448198

  15. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    NASA Astrophysics Data System (ADS)

    Raffar, Nur Izzati Abdul; Rahman, Nadhratul Nur Ain Abdul; Alrozi, Rasyidah; Senusi, Faraziehan; Chang, Siu Hua

    2015-05-01

    Biosorption of copper ion using treated and untreated immobilized Saccharomyces cerevisiae from aqueous solution was investigate in this study. S.cerevisiae has been choosing as biosorbent due to low cost, easy and continuously available from various industries. In this study, the ability of treated and untreated immobilized S.cerevisiae in removing copper ion influence by the effect of pH solution, and initial concentration of copper ion with contact time. Besides, adsorption isotherm and kinetic model also studied. The result indicated that the copper ion uptake on treated and untreated immobilized S.cerevisiae was increased with increasing of contact time and initial concentration of copper ion. The optimum pH for copper ion uptake on untreated and treated immobilized S.cerevisiae at 4 and 6. From the data obtained of copper ion uptake, the adsorption isotherm was fitted well by Freundlich model for treated immobilized S.cerevisiae and Langmuir model for untreated immobilized S.cerevisiae according to high correlation coefficient. Meanwhile, the pseudo second order was described as suitable model present according to high correlation coefficient. Since the application of biosorption process has been received more attention from numerous researchers as a potential process to be applied in the industry, future study will be conducted to investigate the potential of immobilized S.cerevisiae in continuous process.

  16. The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor.

    PubMed Central

    Donald, R G; Schindler, U; Batschauer, A; Cashmore, A R

    1990-01-01

    G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter. Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2161333

  17. Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

    PubMed Central

    Thomas, D; Surdin-Kerjan, Y

    1997-01-01

    Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases

  18. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    PubMed Central

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property. PMID:23383298

  19. Post-Transcriptional Regulation of Iron Homeostasis in Saccharomyces cerevisiae

    PubMed Central

    Martínez-Pastor, María Teresa; de Llanos, Rosa; Romero, Antonia María; Puig, Sergi

    2013-01-01

    Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in a wide variety of biological processes. Recent studies in Saccharomyces cerevisiae have shown that in response to iron deficiency, an RNA-binding protein denoted Cth2 coordinates a global metabolic rearrangement that aims to optimize iron utilization. The Cth2 protein contains two Cx8Cx5Cx3H tandem zinc fingers (TZFs) that specifically bind to adenosine/uridine-rich elements within the 3′ untranslated region of many mRNAs to promote their degradation. The Cth2 protein shuttles between the nucleus and the cytoplasm. Once inside the nucleus, Cth2 binds target mRNAs and stimulates alternative 3′ end processing. A Cth2/mRNA-containing complex is required for export to the cytoplasm, where the mRNA is degraded by the 5′ to 3′ degradation pathway. This post-transcriptional regulatory mechanism limits iron utilization in nonessential pathways and activates essential iron-dependent enzymes such as ribonucleotide reductase, which is required for DNA synthesis and repair. Recent findings indicate that the TZF-containing tristetraprolin protein also functions in modulating human iron homeostasis. Elevated iron concentrations can also be detrimental for cells. The Rnt1 RNase III exonuclease protects cells from excess iron by promoting the degradation of a subset of the Fe acquisition system when iron levels rise. PMID:23903042

  20. Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.

    PubMed

    Verdone, L; Camilloni, G; Di Mauro, E; Caserta, M

    1996-05-01

    We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. PMID:8628264

  1. Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption.

    PubMed

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R H; Jeffries, Thomas W; Olsson, Lisbeth; Nielsen, Jens

    2012-08-01

    Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source. PMID:22487265

  2. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    PubMed

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  3. Modulation of efficiency of translation termination in Saccharomyces cerevisiae

    PubMed Central

    Nizhnikov, Anton A; Antonets, Kirill S; Inge-Vechtomov, Sergey G; Derkatch, Irina L

    2014-01-01

    Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy. PMID:25486049

  4. Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension.

    PubMed

    Alcalde, Miguel

    2010-01-01

    A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols. PMID:20676972

  5. The reference genome sequence of Saccharomyces cerevisiae: then and now.

    PubMed

    Engel, Stacia R; Dietrich, Fred S; Fisk, Dianna G; Binkley, Gail; Balakrishnan, Rama; Costanzo, Maria C; Dwight, Selina S; Hitz, Benjamin C; Karra, Kalpana; Nash, Robert S; Weng, Shuai; Wong, Edith D; Lloyd, Paul; Skrzypek, Marek S; Miyasato, Stuart R; Simison, Matt; Cherry, J Michael

    2014-03-01

    The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called "S288C 2010," was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science. PMID:24374639

  6. Saccharomyces cerevisiae: a sexy yeast with a prion problem.

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

    Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23-46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae. PMID:23764836

  7. The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now

    PubMed Central

    Engel, Stacia R.; Dietrich, Fred S.; Fisk, Dianna G.; Binkley, Gail; Balakrishnan, Rama; Costanzo, Maria C.; Dwight, Selina S.; Hitz, Benjamin C.; Karra, Kalpana; Nash, Robert S.; Weng, Shuai; Wong, Edith D.; Lloyd, Paul; Skrzypek, Marek S.; Miyasato, Stuart R.; Simison, Matt; Cherry, J. Michael

    2014-01-01

    The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called “S288C 2010,” was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science. PMID:24374639

  8. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

    2015-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

  9. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae.

    PubMed

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

  10. Tor1 regulates protein solubility in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Rardin, Matthew J.; Czerwieniec, Gregg; Evani, Uday S.; Reis-Rodrigues, Pedro; Lithgow, Gordon J.; Mooney, Sean D.; Gibson, Bradford W.; Hughes, Robert E.

    2012-01-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase. PMID:23097491

  11. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    PubMed Central

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

  12. Transcriptional regulatory network shapes the genome structure of Saccharomyces cerevisiae

    PubMed Central

    Li, Songling; Heermann, Dieter W.

    2013-01-01

    Among cellular processes gene transcription is central. More and more evidence is mounting that transcription is tightly connected with the spatial organization of the chromosomes. Spatial proximity of genes sharing transcriptional machinery is one of the consequences of this organization. Motivated by information on the physical relationship among genes identified via chromosomal conformation capture methods, we complement the spatial organization with the idea that genes under similar transcription factor control, but possible scattered throughout the genome, might be in physically proximity to facilitate the access of their commonly used transcription factors. Unlike the transcription factory model, “interacting” genes in our “Gene Proximity Model” are not necessarily immediate physical neighbors but are in spatial proximity. Considering the stochastic nature of TF-promoter binding, this local condensation mechanism could serve as a tie to recruit co-regulated genes to guarantee the swiftness of biological reactions. We tested this idea with a simple eukaryotic organism, Saccharomyces cerevisiae. Chromosomal interaction patterns and folding behavior generated by our model re-construct those obtained from experiments. We show that the transcriptional regulatory network has a close linkage with the genome organization in budding yeast, which is fundamental and instrumental to later studies on other more complex eukaryotes. PMID:23674068

  13. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific

    PubMed Central

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

  14. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific.

    PubMed

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

  15. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  16. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    PubMed Central

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  17. Saccharomyces boulardii

    MedlinePlus

    ... believed to be a strain of Saccharomyces cerevisiae (baker's yeast). Saccharomyces boulardii is used as medicine. Saccharomyces boulardii ... Hansen CBS 5926), Probiotic, Probiotique, Saccharomyces, Saccharomyces boulardii, Saccharomyces Cerevisiae, S. Boulardii.

  18. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

    The budding yeast Saccharomyces cerevisiae has two alternative mating types designated MATa and MATα. These are distinguished by about 700 bp of unique sequences, Ya or Yα, including divergent promoter sequences and part of the open reading frames of genes that regulate mating phenotype. Homothallic budding yeast, carrying an active HO endonuclease gene, HO, can switch mating type through a recombination process known as gene conversion, in which a site-specific double-strand break (DSB) created immediately adjacent to the Y region results in replacement of the Y sequences with a copy of the opposite mating type information, which is harbored in one of two heterochromatic donor loci, HMLα or HMRa. HO gene expression is tightly regulated to ensure that only half of the cells in a lineage switch to the opposite MAT allele, thus promoting conjugation and diploid formation. Study of the silencing of these loci has provided a great deal of information about the role of the Sir2 histone deacetylase and its associated Sir3 and Sir4 proteins in creating heterochromatic regions. MAT switching has been examined in great detail to learn about the steps in homologous recombination. MAT switching is remarkably directional, with MATa recombining preferentially with HMLα and MATα using HMRa. Donor preference is controlled by a cis-acting recombination enhancer located near HML. RE is turned off in MATα cells but in MATa binds multiple copies of the Fkh1 transcription factor whose forkhead-associated phosphothreonine binding domain localizes at the DSB, bringing HML into conjunction with MATa. PMID:26104712

  19. A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

    PubMed Central

    Fang, Fang; Salmon, Kirsty; Shen, Michael W. Y.; Aeling, Kimberly A.; Ito, Elaine; Irwin, Becky; Tran, Uyen Phuong C.; Hatfield, G. Wesley; Da Silva, Nancy A.; Sandmeyer, Suzanne

    2011-01-01

    A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 μ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering. PMID:20936606

  20. Saccharomyces cerevisiae: a nomadic yeast with no niche?

    PubMed Central

    Goddard, Matthew R.; Greig, Duncan

    2015-01-01

    Different species are usually thought to have specific adaptations, which allow them to occupy different ecological niches. But recent neutral ecology theory suggests that species diversity can simply be the result of random sampling, due to finite population sizes and limited dispersal. Neutral models predict that species are not necessarily adapted to specific niches, but are functionally equivalent across a range of habitats. Here, we evaluate the ecology of Saccharomyces cerevisiae, one of the most important microbial species in human history. The artificial collection, concentration and fermentation of large volumes of fruit for alcohol production produce an environment in which S. cerevisiae thrives, and therefore it is assumed that fruit is the ecological niche that S. cerevisiae inhabits and has adapted to. We find very little direct evidence that S. cerevisiae is adapted to fruit, or indeed to any other specific niche. We propose instead a neutral nomad model for S. cerevisiae, which we believe should be used as the starting hypothesis in attempting to unravel the ecology of this important microbe. PMID:25725024

  1. Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.

    PubMed

    Mormeneo, María; Pastor, Fi Javier; Zueco, Jesús

    2012-01-01

    The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase. PMID:21701899

  2. In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae.

    PubMed Central

    Clark-Walker, G D

    1989-01-01

    A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps. Images PMID:2682661

  3. A Saccharomyces cerevisiae Internet protein resource now available.

    PubMed

    Latter, G I; Boutell, T; Monardo, P J; Kobayashi, R; Futcher, B; Mclaughlin, C S; Garrels, J I

    1995-07-01

    The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World-Wide Web (WWW) server (URL http:@siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other. PMID:7498160

  4. Purification of fluorescently labeled Saccharomyces cerevisiae Spindle Pole Bodies

    PubMed Central

    Davis, Trisha N.

    2016-01-01

    Centrosomes are components of the mitotic spindle responsible for organizing microtubules and establishing a bipolar spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the spindle pole body, a highly organized tri-laminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled spindle pole bodes from S. cerevisiae. Spindle pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation. This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast spindle pole bodies. The genetic flexibility of this technique allows for the study of spindle pole body mutants as well as the study of spindle pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique makes it possible to study spindle pole bodies using a variety of biochemical, biophysical, and microscopic techniques. PMID:27193850

  5. Advanced biofuel production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Siewers, Verena; Nielsen, Jens

    2013-06-01

    Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels. PMID:23628723

  6. Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae.

    PubMed Central

    Kirchman, P A; Kim, S; Lai, C Y; Jazwinski, S M

    1999-01-01

    Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae. In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence. Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S. cerevisiae strains. Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity. RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity. The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability. PMID:10224252

  7. Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

    SciTech Connect

    Batt, C.A.; Carvallo, S.; Easson, D.D.; Akedo, M.; Sinskey, A.J.

    1986-04-01

    Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisie as compared to Candida utilis. In vivo conversion of /sup 14/C-xylose in S. cerevisiage is demonstrated and xylitol is detected, although no significant levels of any other /sup 14/C-labeled metabolites (e.g., ethanol) are observed. 22 references.

  8. ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Vishnu Varthini, Lakshmanaperumal; Selvaraju, Kandasamy; Srinivasan, Malathi; Nachiappan, Vasanthi

    2015-01-01

    Lipid metabolism is extensively studied in Saccharomyces cerevisiae. Here, we report that revertant of glycogen synthase kinase mutation-1 (Rog1p) possesses monoacylglycerol (MAG) lipase activity in S. cerevisiae. The lipase activity of Rog1p was confirmed in two ways: through analysis of a strain with a double deletion of ROG1 and monoglyceride lipase YJU3 (yju3Δrog1Δ) and by site-directed mutagenesis of the ROG1 lipase motif (GXSXG). Rog1p is localized in both the cytosol and the nucleus. Overexpression of ROG1 in a ROG1-deficient strain resulted in an accumulation of reactive oxygen species. These results suggest that Rog1p is a MAG lipase that regulates lipid homeostasis. PMID:25433290

  9. Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate

    SciTech Connect

    Raugei, G.; Modesti, A.; Magherini, F.

    1996-06-01

    Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes. 20 refs., 5 figs.

  10. Immobilized cell cross-flow reactor. [Saccharomyces cerevisiae

    SciTech Connect

    Chotani, G.K.; Constantinides, A.

    1984-01-01

    A cross-current flow reactor was operated using sodium alginate gel entrapped yeast cells (Saccharomyces cerevisiae) under growth conditions. Micron-sized silica, incorporated into the biocatalyst particles (1 mm mean diameter) improved mechanical strength and internal surface adhesion. The process showed decreased productivity and stability at 35/sup 0/C compared to the normal study done at 30/sup 0/C. The increased number of cross flows diminish the product inhibition effect. The residence time distribution shows that the cross-flow bioreactor system can be approximated to either a train of backmixed fermentors in series or a plug flow fermentor with moderate axial dispersion.

  11. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

    PubMed Central

    Ogden, R C; Lee, M C; Knapp, G

    1984-01-01

    The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease. Images PMID:6096826

  12. SACCHAROMYCES CEREVISIAE Recessive Suppressor That Circumvents Phosphatidylserine Deficiency

    PubMed Central

    Atkinson, Katharine D.

    1984-01-01

    Phenotypic reversion of six independent Saccharomyces cerevisiae cho1 mutants was shown to be due predominantly to mutation of an unlinked gene, eam1. The eam1 gene was located very close to ino1 on chromosome X by meiotic tetrad analysis. Recessive eam1 mutations did not correct the primary cho1 defect in phosphatidylserine synthesis but made endogenous ethanolamine available for sustained nitrogenous phospholipid synthesis. A novel biochemical contribution to nitrogenous lipid synthesis is indicated by the eam1 mutants. PMID:17246236

  13. Mutants of the Formyltetrahydrofolate Interconversion Pathway of SACCHAROMYCES CEREVISIAE

    PubMed Central

    McKenzie, K. Q.; Jones, Elizabeth W.

    1977-01-01

    Thirteen mutants of Saccharomyces cerevisiae that lack one or more of the three enzyme activities of the pathway for interconversion of tetrahydrofolate coenzymes at the formate level of oxidation have been isolated. They do not require adenine. All fail to complement mutations in the ade3 locus. Mutations that greatly reduce activity for one enzyme also reduce activity for the other two interconversion enzymes. The three enzyme activities cochromatograph on TEAE-cellulose columns. A mutation that eliminates synthetase activity also alters the chromatographic behavior of the remaining cyclohydrolase and dehydrogenase activities. It is suggested that the three activities reside in an enzyme complex encoded by the ade3 locus. PMID:328341

  14. Use of bimolecular fluorescence complementation in yeast Saccharomyces cerevisiae.

    PubMed

    Skarp, Kari-Pekka; Zhao, Xueqiang; Weber, Marion; Jantti, Jussi

    2008-01-01

    Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. Bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for use of BiFC in the yeast Saccharomyces cerevisiae. PMID:19066026

  15. RNAi-Assisted Genome Evolution (RAGE) in Saccharomyces cerevisiae.

    PubMed

    Si, Tong; Zhao, Huimin

    2016-01-01

    RNA interference (RNAi)-assisted genome evolution (RAGE) applies directed evolution principles to engineer Saccharomyces cerevisiae genomes. Here, we use acetic acid tolerance as a target trait to describe the key steps of RAGE. Briefly, iterative cycles of RNAi screening are performed to accumulate multiplex knockdown modifications, enabling directed evolution of the yeast genome and continuous improvement of a target phenotype. Detailed protocols are provided on the reconstitution of RNAi machinery, creation of genome-wide RNAi libraries, identification and integration of beneficial knockdown cassettes, and repeated RAGE cycles. PMID:27581294

  16. Isobutanol production from D-xylose by recombinant Saccharomyces cerevisiae.

    PubMed

    Brat, Dawid; Boles, Eckhard

    2013-03-01

    Simultaneous overexpression of an optimized, cytosolically localized valine biosynthesis pathway together with overexpression of xylose isomerase XylA from Clostridium phytofermentans, transaldolase Tal1 and xylulokinase Xks1 enabled recombinant Saccharomyces cerevisiae cells to complement the valine auxotrophy of ilv2,3,5 triple deletion mutants for growth on D-xylose as the sole carbon source. Moreover, after additional overexpression of ketoacid decarboxylase Aro10 and alcohol dehydrogenase Adh2, the cells were able to ferment D-xylose directly to isobutanol. PMID:23279585

  17. Differential repair of UV damage in Saccharomyces cerevisiae.

    PubMed Central

    Terleth, C; van Sluis, C A; van de Putte, P

    1989-01-01

    Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed. Images PMID:2664708

  18. Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

    PubMed

    Marques, Wesley Leoricy; Raghavendran, Vijayendran; Stambuk, Boris Ugarte; Gombert, Andreas Karoly

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering. PMID:26658003

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

    PubMed

    Naumov, G I; Naumova, E S; Michels, C A

    1994-03-01

    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. PMID:8005435

  20. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions. PMID:27221740

  1. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

    PubMed Central

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S.; Flick, Robert; Wolf, Yuri I.; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D.; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M.; Koonin, Eugene V.; Yakunin, Alexander F.

    2015-01-01

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. PMID:26071590

  2. Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods

    PubMed Central

    Tofalo, Rosanna; Perpetuini, Giorgia; Schirone, Maria; Fasoli, Giuseppe; Aguzzi, Irene; Corsetti, Aldo; Suzzi, Giovanna

    2013-01-01

    Biogeography is the descriptive and explanatory study of spatial patterns and processes involved in the distribution of biodiversity. Without biogeography, it would be difficult to study the diversity of microorganisms because there would be no way to visualize patterns in variation. Saccharomyces cerevisiae, “the wine yeast,” is the most important species involved in alcoholic fermentation, and in vineyard ecosystems, it follows the principle of “everything is everywhere.” Agricultural practices such as farming (organic versus conventional) and floor management systems have selected different populations within this species that are phylogenetically distinct. In fact, recent ecological and geographic studies highlighted that unique strains are associated with particular grape varieties in specific geographical locations. These studies also highlighted that significant diversity and regional character, or ‘terroir,’ have been introduced into the winemaking process via this association. This diversity of wild strains preserves typicity, the high quality, and the unique flavor of wines. Recently, different molecular methods were developed to study population dynamics of S. cerevisiae strains in both vineyards and wineries. In this review, we will provide an update on the current molecular methods used to reveal the geographical distribution of S. cerevisiae wine yeast. PMID:23805132

  3. Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.

    PubMed Central

    Makarow, M

    1985-01-01

    When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells. Images Fig. 2. Fig. 4. PMID:2992948

  4. [Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].

    PubMed

    Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli

    2014-08-01

    β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25507473

  5. [Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].

    PubMed

    Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli

    2014-08-01

    β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25423750

  6. Copper oxide nanoparticles inhibit the metabolic activity of Saccharomyces cerevisiae.

    PubMed

    Mashock, Michael J; Kappell, Anthony D; Hallaj, Nadia; Hristova, Krassimira R

    2016-01-01

    Copper oxide nanoparticles (CuO NPs) are used increasingly in industrial applications and consumer products and thus may pose risk to human and environmental health. The interaction of CuO NPs with complex media and the impact on cell metabolism when exposed to sublethal concentrations are largely unknown. In the present study, the short-term effects of 2 different sized manufactured CuO NPs on metabolic activity of Saccharomyces cerevisiae were studied. The role of released Cu(2+) during dissolution of NPs in the growth media and the CuO nanostructure were considered. Characterization showed that the 28 nm and 64 nm CuO NPs used in the present study have different primary diameter, similar hydrodynamic diameter, and significantly different concentrations of dissolved Cu(2+) ions in the growth media released from the same initial NP mass. Exposures to CuO NPs or the released Cu(2+) fraction, at doses that do not have impact on cell viability, showed significant inhibition on S. cerevisiae cellular metabolic activity. A greater CuO NP effect on the metabolic activity of S. cerevisiae growth under respiring conditions was observed. Under the tested conditions the observed metabolic inhibition from the NPs was not explained fully by the released Cu ions from the dissolving NPs. PMID:26178758

  7. Membrane trafficking in the yeast Saccharomyces cerevisiae model.

    PubMed

    Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L; Friant, Sylvie

    2015-01-01

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes. PMID:25584613

  8. Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae.

    PubMed

    Citterio, Barbara; Albertini, Maria Cristina; Ghibelli, Lina; Falcieri, Elisabetta; Battistelli, Michela; Canonico, Barbara; Rocchi, Marco B L; Teodori, Laura; Ciani, Maurizio; Piatti, Elena

    2015-08-01

    In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection. PMID:25868793

  9. Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

    SciTech Connect

    Yannai, S.; Berdicevsky, I.; Duek, L. )

    1991-01-01

    Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 {mu}g of Hg (as HgCl{sub 2}) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28{degree}C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows: (1) C. albicans was the more mercury-resistant species, but both yeast species failed to grown in the media containing 0.75 {mu}g of Hg per ml.; (2) The amounts of organomercury produced by the two species were proportional to the amount of HgCl{sub 2} added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae; (3) The amounts of elemental Hg produced were inversely proportional to the HgCl{sub 2} level added in the case of S. cerevisiae but were all similar in the case of C. albicans;and (4) Neither organomercury nor elemental Hg was produced in any of the control media.

  10. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    PubMed Central

    Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L.; Friant, Sylvie

    2015-01-01

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes. PMID:25584613

  11. Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production.

    PubMed

    Chen, Binbin; Lee, Dong-Yup; Chang, Matthew Wook

    2015-09-01

    Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae. PMID:26164646

  12. Microencapsulation of Saccharomyces cerevisiae and its evaluation to protect in simulated gastric conditions

    PubMed Central

    Ghorbani-Choboghlo, Hassan; Zahraei-Salehi, Taghi; Ashrafi-Helan, Javad; Yahyaraeyat, Ramak; Pourjafar, Hadi; Nikaein, Donya; Balal, Asad; Khosravi, Ali-Reza

    2015-01-01

    Background and Objectives: Probiotic yeasts are used in production of functional foods and pharmaceutical products. They play an important role in promoting and maintaining human health. Until now, little work has been published on improving the survival of Saccharomyces in stimulated gastrointestinal condition. Material and Methods: In this study the exposure of the yeast in the capsulate and free forms to artificial gastrointestinal conditions was assessed and the number of viable Saccharomyces cerevisiae cells during 0 to 120 mines in these conditions was evaluated by a pour plate method using sabouraud dextrose agar. Results: Results showed the shape of the beads was generally spherical, sometimes elliptical with a mean diameter of about 50–90 μm. Also count of viable probiotic cells obtained for all the microcapsules were above the recommended levels for a probiotic food. Also decrease of approximately 4 logs was noted in the number of free cells after 2 h of incubation at pH 2 and 8, when compared to decreases of about 2 logs in the all microencapsulated S. cerevisiae under similar conditions. Conclusion: It is concluded that microencapsulation process was significantly able to increase the survival rate of Saccharomyces in a simulated gastrointestinal condition (p<0.05).. PMID:26885335

  13. Identification of Novel Knockout Targets for Improving Terpenoids Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8–10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids. PMID:25386654

  14. The Mitochondrial RNA Landscape of Saccharomyces cerevisiae

    PubMed Central

    Turk, Edward M.; Das, Vaijayanti; Seibert, Ryan D.; Andrulis, Erik D.

    2013-01-01

    Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay. PMID:24143261

  15. The mitochondrial RNA landscape of Saccharomyces cerevisiae.

    PubMed

    Turk, Edward M; Das, Vaijayanti; Seibert, Ryan D; Andrulis, Erik D

    2013-01-01

    Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay. PMID:24143261

  16. Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

    PubMed Central

    Curry, Claudia; Gilkes, Neil; O'Neill, Gary; Miller, Robert C.; Skipper, Nigel

    1988-01-01

    We used the yeast MEL1 gene for secreted α-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi β-1,4-exoglucanase was inserted into one cartridge to create a fusion of the α-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-β-d-cellobiose, and p-nitrophenyl-β-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical Km and pH optimum but to be more thermostable. Images PMID:16347562

  17. Determinants of Swe1p Degradation in Saccharomyces cerevisiae

    PubMed Central

    McMillan, John N.; Theesfeld, Chandra L.; Harrison, Jacob C.; Bardes, Elaine S. G.; Lew, Daniel J.

    2002-01-01

    Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCFMet30 acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCFMet30) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. PMID:12388757

  18. Comparative proteomic analysis of Saccharomyces cerevisiae under different nitrogen sources.

    PubMed

    Zhao, Shaohui; Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-04-14

    In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions. PMID:24530623

  19. Production of natural products through metabolic engineering of Saccharomyces cerevisiae.

    PubMed

    Krivoruchko, Anastasia; Nielsen, Jens

    2015-12-01

    Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways of these compounds in organisms that are more suitable for industrial production. Recent years have seen developments in both the discovery of various biosynthetic pathways, as well as development of metabolic engineering tools that allow reconstruction of complex pathways in microorganisms. In the present review we discuss recent advances in reconstruction of the biosynthetic pathways of various high-value products in the yeast Saccharomyces cerevisiae, a commonly used industrial microorganism. Key achievements in the production of different isoprenoids, aromatics and polyketides are presented and the metabolic engineering strategies underlying these accomplishments are discussed. PMID:25544013

  20. Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

    SciTech Connect

    Hayashi, Naoyuki Kobayashi, Masahiko; Shimizu, Hiroko; Yamamoto, Ken-ichi; Murakami, Seishi; Nishimoto, Takeharu

    2007-11-23

    The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-{delta}2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a high dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.

  1. Bioaccumulation of cadmium by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae.

    PubMed

    Li, Chunsheng; Jiang, Wei; Ma, Ning; Zhu, Yinglian; Dong, Xiaoyan; Wang, Dongfeng; Meng, Xianghong; Xu, Ying

    2014-03-01

    Bioaccumulation via growing cells is a potential technique for heavy metal removal from food materials. The cadmium bioaccumulation characteristics by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae were investigated. Z. rouxii displayed powerful cadmium removal ability at low cadmium concentrations, which mainly depended on the intracellular cadmium bioaccumulation. The percentage of intracellular cadmium bioaccumulation of both yeasts obviously decreased with the increase of initial biomass and cadmium concentrations. Low pH and elevated concentrations of zinc and copper significantly decreased the intracellular cadmium bioaccumulation of both yeasts but improved the cadmium tolerance and the cell-surface cadmium bioaccumulation of Z. rouxii. Cadmium removal of Z. rouxii was improved by zinc and copper conditionally. Z. rouxii that possessed more powerful cadmium tolerance and removal ability at low pH and high concentration of competing ions can be developed into a potential cadmium removal agent using in complex food environment in future. PMID:24440489

  2. MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae.

    PubMed

    Conrad, Michael N; Lee, Chih-Ying; Wilkerson, Joseph L; Dresser, Michael E

    2007-05-22

    In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1 Delta-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements. PMID:17495028

  3. The Influence of Microgravity on Invasive Growth in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Van Mulders, Sebastiaan E.; Stassen, Catherine; Daenen, Luk; Devreese, Bart; Siewers, Verena; van Eijsden, Rudy G. E.; Nielsen, Jens; Delvaux, Freddy R.; Willaert, Ronnie

    2011-01-01

    This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

  4. Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae.

    PubMed

    Li, C; Li, J; Wang, G; Li, X

    2016-06-01

    Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l(-1) ; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up-regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform. PMID:26743771

  5. Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.

    PubMed

    Altmann, M; Blum, S; Pelletier, J; Sonenberg, N; Wilson, T M; Trachsel, H

    1990-08-27

    Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal. PMID:2169890

  6. Availability of substratum enhances ethanol production in Saccharomyces cerevisiae.

    PubMed

    Sankh, Santosh N; Arvindekar, Akalpita U

    2004-12-01

    Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30-40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40-50 mg g (-1) dry yeast while intracellular glycogen decreased by 10-12 mg g(-1) dry yeast in cells grown in presence of substratum. PMID:15672221

  7. Yap1: a DNA damage responder in Saccharomyces cerevisiae.

    PubMed

    Rowe, Lori A; Degtyareva, Natalya; Doetsch, Paul W

    2012-04-01

    Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents. PMID:22433435

  8. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    PubMed

    D'Amore, T; Panchal, C J; Stewart, G G

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures. PMID:3278685

  9. Mutants of Saccharomyces cerevisiae with defective vacuolar function

    SciTech Connect

    Kitamoto, K.; Yoshizawa, K.; Ohsumi, Y.; Anraku, Y.

    1988-06-01

    Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca/sup 2 +/, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker ..cap alpha..-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.

  10. Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

    PubMed Central

    Williams, J S; Eckdahl, T T; Anderson, J N

    1988-01-01

    Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function. Images PMID:3043195

  11. Identity elements of Saccharomyces cerevisiae tRNA(His).

    PubMed Central

    Nameki, N; Asahara, H; Shimizu, M; Okada, N; Himeno, H

    1995-01-01

    Recognition of tRNA(His) by Saccharomyces cerevisiae histidyl-tRNA synthetase was studied using in vitro transcripts. Histidine tRNA is unique in possessing an extra nucleotide, G-1, at the 5' end. Mutation studies indicate that this irregular secondary structure at the end of the acceptor stem is important for aminoacylation with histidine, while the requirement of either base of this extra base pair is smaller than that in Escherichia coli. The anticodon was also found to be required for histidylation. The regions involved in histidylation are essentially the same as those in E.coli, whereas the proportion of the contributions of the two portions distant from each other, the anticodon and the end of the acceptor stem, makes a substantial difference between the two systems. PMID:7885835

  12. Hormetic Effect of H2O2 in Saccharomyces cerevisiae

    PubMed Central

    Valishkevych, Bohdana V.

    2016-01-01

    In this study, we investigated the relationship between target of rapamycin (TOR) and H2O2-induced hormetic response in the budding yeast Saccharomyces cerevisiae grown on glucose or fructose. In general, our data suggest that: (1) hydrogen peroxide (H2O2) induces hormesis in a TOR-dependent manner; (2) the H2O2-induced hormetic dose–response in yeast depends on the type of carbohydrate in growth medium; (3) the concentration-dependent effect of H2O2 on yeast colony growth positively correlates with the activity of glutathione reductase that suggests the enzyme involvement in the H2O2-induced hormetic response; and (4) both TOR1 and TOR2 are involved in the reciprocal regulation of the activity of glucose-6-phosphate dehydrogenase and glyoxalase 1. PMID:27099601

  13. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

    PubMed

    Gallone, Brigida; Steensels, Jan; Prahl, Troels; Soriaga, Leah; Saels, Veerle; Herrera-Malaver, Beatriz; Merlevede, Adriaan; Roncoroni, Miguel; Voordeckers, Karin; Miraglia, Loren; Teiling, Clotilde; Steffy, Brian; Taylor, Maryann; Schwartz, Ariel; Richardson, Toby; White, Christopher; Baele, Guy; Maere, Steven; Verstrepen, Kevin J

    2016-09-01

    Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP. PMID:27610566

  14. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  15. Exposure to benzene metabolites causes oxidative damage in Saccharomyces cerevisiae.

    PubMed

    Raj, Abhishek; Nachiappan, Vasanthi

    2016-06-01

    Hydroquinone (HQ) and benzoquinone (BQ) are known benzene metabolites that form reactive intermediates such as reactive oxygen species (ROS). This study attempts to understand the effect of benzene metabolites (HQ and BQ) on the antioxidant status, cell morphology, ROS levels and lipid alterations in the yeast Saccharomyces cerevisiae. There was a reduction in the growth pattern of wild-type cells exposed to HQ/BQ. Exposure of yeast cells to benzene metabolites increased the activity of the anti-oxidant enzymes catalase, superoxide dismutase and glutathione peroxidase but lead to a decrease in ascorbic acid and reduced glutathione. Increased triglyceride level and decreased phospholipid levels were observed with exposure to HQ and BQ. These results suggest that the enzymatic antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumptively, these enzymes are essential for scavenging the pro-oxidant effects of benzene metabolites. PMID:27016252

  16. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    NASA Astrophysics Data System (ADS)

    Shibata, M.; Torigoe, M.; Matsumoto, Y.; Yamamoto, M.; Takizawa, N.; Hada, Y.; Mori, Y.; Takarabe, K.; Ono, F.

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  17. Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1.

    PubMed

    Busygina, Valeria; Gaines, William A; Xu, Yuanyuan; Kwon, Youngho; Williams, Gareth J; Lin, Sheng-Wei; Chang, Hao-Yen; Chi, Peter; Wang, Hong-Wei; Sung, Patrick

    2013-09-01

    The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase. PMID:23769192

  18. Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock.

    PubMed

    Vicedo, Esmeralda; Gasik, Zofia; Dong, Yu-An; Goldberg, Tatyana; Rost, Burkhard

    2015-01-01

    Recent experiments established that a culture of Saccharomyces cerevisiae (baker's yeast) survives sudden high temperatures by specifically duplicating the entire chromosome III and two chromosomal fragments (from IV and XII). Heat shock proteins (HSPs) are not significantly over-abundant in the duplication. In contrast, we suggest a simple algorithm to " postdict " the experimental results: Find a small enough chromosome with minimal protein disorder and duplicate this region. This algorithm largely explains all observed duplications. In particular, all regions duplicated in the experiment reduced the overall content of protein disorder. The differential analysis of the functional makeup of the duplication remained inconclusive. Gene Ontology (GO) enrichment suggested over-representation in processes related to reproduction and nutrient uptake. Analyzing the protein-protein interaction network (PPI) revealed that few network-central proteins were duplicated. The predictive hypothesis hinges upon the concept of reducing proteins with long regions of disorder in order to become less sensitive to heat shock attack. PMID:26673203

  19. Higher-order structure of Saccharomyces cerevisiae chromatin

    SciTech Connect

    Lowary, P.T.; Widom, J. )

    1989-11-01

    We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast killer virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.

  20. Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Pelling, Andrew E.; Sehati, Sadaf; Gralla, Edith B.; Valentine, Joan S.; Gimzewski, James K.

    2004-08-01

    We demonstrate that the cell wall of living Saccharomyces cerevisiae (baker's yeast) exhibits local temperature-dependent nanomechanical motion at characteristic frequencies. The periodic motions in the range of 0.8 to 1.6 kHz with amplitudes of ~3 nm were measured using the cantilever of an atomic force microscope (AFM). Exposure of the cells to a metabolic inhibitor causes the periodic motion to cease. From the strong frequency dependence on temperature, we derive an activation energy of 58 kJ/mol, which is consistent with the cell's metabolism involving molecular motors such as kinesin, dynein, and myosin. The magnitude of the forces observed (~10 nN) suggests concerted nanomechanical activity is operative in the cell.

  1. Construction of a flocculent Saccharomyces cerevisiae fermenting lactose.

    PubMed

    Domingues, L; Teixeira, J A; Lima, N

    1999-05-01

    A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. PMID:10390820

  2. Characterization of Encapsulated Berberine in Yeast Cells of Saccharomyces cerevisiae

    PubMed Central

    Salari, Roshanak; Rajabi, Omid; Khashyarmanesh, Zahra; Fathi Najafi, Mohsen; Fazly Bazzaz, BiBi Sedigheh

    2015-01-01

    Berberine was loaded in yeast cells of Saccharomyces cerevisiaeas a novel pharmaceutical carrier to improve the treatment ofmany diseases. The yeast-encapsulated active materialsshowedhigh stability and bioavailability due to the enhanced solubility and sustained releasing. In this study, different characteristics of prepared berberine loaded yeast cells (loading capacity, release kinetic order, MIC and stability) were evaluatedby different analytical methods (fluorescence spectroscopy, HPLC and SEM).The loading capacity was about 78% ± 0.6%.Berberine release patterns of microcapsules happened in two different stages and followed by zero and first-order kinetic,respectively. About 99% of all active material released during 34 h. MIC was improved by berberine loaded microcapsules in comparison withberberine powder. The microcapsules were completely stable. Berberine loaded Sac. Cerevisiae could be considered as a favorite sustained release drug delivery system. The yeast would be applied as an efficient carrier to improve various properties of different active materials. PMID:26664393

  3. Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

    PubMed Central

    Liu, Ling-ling; Jia, Bo; Zhao, Fang; Huang, Wei-dong; Zhan, Ji-cheng

    2015-01-01

    At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress. PMID:26030864

  4. Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae.

    PubMed

    Trabalzini, Lorenza; Paffetti, Alessandro; Ferro, Elisa; Scaloni, Andrea; Talamo, Fabio; Millucci, Lia; Martelli, Paola; Santucci, Annalisa

    2003-12-01

    Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines. PMID:15141481

  5. Water treatment process and system for metals removal using Saccharomyces cerevisiae

    DOEpatents

    Krauter, Paula A. W.; Krauter, Gordon W.

    2002-01-01

    A process and a system for removal of metals from ground water or from soil by bioreducing or bioaccumulating the metals using metal tolerant microorganisms Saccharomyces cerevisiae. Saccharomyces cerevisiae is tolerant to the metals, able to bioreduce the metals to the less toxic state and to accumulate them. The process and the system is useful for removal or substantial reduction of levels of chromium, molybdenum, cobalt, zinc, nickel, calcium, strontium, mercury and copper in water.

  6. The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

    PubMed Central

    Cameron, D R; Cooper, D G; Neufeld, R J

    1988-01-01

    The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier. PMID:3046488

  7. Systems biology of GAL regulon in Saccharomyces cerevisiae.

    PubMed

    Pannala, Venkat Reddy; Bhat, Paike Jayadeva; Bhartiya, Sharad; Venkatesh, K V

    2010-01-01

    Evolutionary success of an organism depends on its ability to express or adapt to constantly changing environmental conditions. Saccharomyces cerevisiae has evolved an elaborate genetic circuit to regulate the expression of galactose-metabolizing enzymes in the presence of galactose but in the absence of glucose. The circuit possesses molecular mechanisms such as multiple binding sites, cooperativity, autoregulation, nucleocytoplasmic shuttling, and substrate sensing mechanism. Furthermore, the GAL system consists of two positive (activating) feedback and one negative (repressing) feedback loops. These individual mechanisms, elucidated through experimental approach, can be integrated to obtain a system-wide behavior. Mathematical models in conjunction with guided experiments have demonstrated system-level properties such as ultrasensitivity, memory, noise attenuation, rapid response, and sensitive response arising out of the molecular interactions. These system-level properties allow S. cerevisiae to adapt and grow in a galactose medium under noisy and changing environments. This review focuses on system-level models and properties of the GAL regulon. PMID:20836013

  8. Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.

    PubMed

    Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M

    2009-08-01

    Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production. PMID:19481129

  9. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-01-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940

  10. Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.

    PubMed

    Yu, Xin; Gabriel, Abram

    2004-02-01

    Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair. PMID:15020464

  11. Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Ohta, Erika; Nakayama, Yasumune; Mukai, Yukio; Bamba, Takeshi; Fukusaki, Eiichiro

    2016-04-01

    The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance. PMID:26344121

  12. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

    PubMed Central

    Galibert, F; Alexandraki, D; Baur, A; Boles, E; Chalwatzis, N; Chuat, J C; Coster, F; Cziepluch, C; De Haan, M; Domdey, H; Durand, P; Entian, K D; Gatius, M; Goffeau, A; Grivell, L A; Hennemann, A; Herbert, C J; Heumann, K; Hilger, F; Hollenberg, C P; Huang, M E; Jacq, C; Jauniaux, J C; Katsoulou, C; Karpfinger-Hartl, L

    1996-01-01

    The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome. Images PMID:8641269

  13. Long-chain alkane production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens

    2015-06-01

    In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfd1 and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFD1 together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol. PMID:25545362

  14. Quantifying separation and similarity in a Saccharomyces cerevisiae metapopulation

    PubMed Central

    Knight, Sarah; Goddard, Matthew R

    2015-01-01

    Eukaryotic microbes are key ecosystem drivers; however, we have little theory and few data elucidating the processes influencing their observed population patterns. Here we provide an in-depth quantitative analysis of population separation and similarity in the yeast Saccharomyces cerevisiae with the aim of providing a more detailed account of the population processes occurring in microbes. Over 10 000 individual isolates were collected from native plants, vineyards and spontaneous ferments of fruit from six major regions spanning 1000 km across New Zealand. From these, hundreds of S. cerevisiae genotypes were obtained, and using a suite of analytical methods we provide comprehensive quantitative estimates for both population structure and rates of gene flow or migration. No genetic differentiation was detected within geographic regions, even between populations inhabiting native forests and vineyards. We do, however, reveal a picture of national population structure at scales above ∼100 km with distinctive populations in the more remote Nelson and Central Otago regions primarily contributing to this. In addition, differential degrees of connectivity between regional populations are observed and correlate with the movement of fruit by the New Zealand wine industry. This suggests some anthropogenic influence on these observed population patterns. PMID:25062126

  15. Data on dynamic study of cytoophidia in Saccharomyces cerevisiae.

    PubMed

    Li, Hui; Huang, Yong; Wang, Peng-Ye; Ye, Fangfu; Liu, Ji-Long

    2016-09-01

    The data in this paper are related to the research article entitled "Filamentation of metabolic enzymes in Saccharomyces cerevisiae" Q.J. Shen et al. (2016) [1]. Cytoophidia are filamentous structures discovered in fruit flies (doi:10.1016/S1673-8527(09)60046-1) J.L. Liu (2010) [2], bacteria (doi:10.1038/ncb2087) M. Ingerson-Mahar et al. (2010) [3], yeast (doi:10.1083/jcb.201003001; doi:10.1242/bio.20149613) C. Noree et al. (2010) and J. Zhang, L. Hulme, J.L. Liu (2014) [4], [5] and human cells (doi:10.1371/journal.pone.0029690; doi:10.1016/j.jgg.2011.08.004) K. Chen et al. (2011) and W.C. Carcamo et al. (2011) ( [6], [7]. However, there is little research on the motility of the cytoophidia. Here we selected cytoophidia formed by 6 filament-forming proteins in the budding yeast S. cerevisiae, and performed living-cell imaging of cells expressing the proteins fused with GFP. The dynamic features of the six types of cytoophidia were analyzed. In the data, both raw movies and analysed results of the dynamics of cytoophidia are presented. PMID:27274529

  16. Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae.

    PubMed

    Swinnen, Steve; Ho, Ping-Wei; Klein, Mathias; Nevoigt, Elke

    2016-07-01

    The yeast Saccharomyces cerevisiae generally shows a low natural capability to utilize glycerol as the sole source of carbon, particularly when synthetic medium is used and complex supplements are omitted. Nevertheless, wild type isolates have been identified that show a moderate growth under these conditions. In the current study we made use of intraspecies diversity to identify targets suitable for reverse metabolic engineering of the non-growing laboratory strain CEN.PK113-1A. A genome-wide genetic mapping experiment using pooled-segregant whole-genome sequence analysis was conducted, and one major and several minor genetic loci were identified responsible for the superior glycerol growth phenotype of the previously selected S. cerevisiae strain CBS 6412-13A. Downscaling of the major locus by fine-mapping and reciprocal hemizygosity analysis allowed the parallel identification of two superior alleles (UBR2CBS 6412-13A and SSK1CBS 6412-13A). These alleles together with the previously identified GUT1CBS 6412-13A allele were used to replace the corresponding alleles in the strain CEN.PK113-1A. In this way, glycerol growth could be established reaching a maximum specific growth rate of 0.08h(-1). Further improvement to a maximum specific growth rate of 0.11h(-1) could be achieved by heterologous expression of the glycerol facilitator FPS1 from Cyberlindnera jadinii. PMID:26971668

  17. Genomic Evolution of Saccharomyces cerevisiae under Chinese Rice Wine Fermentation

    PubMed Central

    Li, Yudong; Zhang, Weiping; Zheng, Daoqiong; Zhou, Zhan; Yu, Wenwen; Zhang, Lei; Feng, Lifang; Liang, Xinle; Guan, Wenjun; Zhou, Jingwen; Chen, Jian; Lin, Zhenguo

    2014-01-01

    Rice wine fermentation represents a unique environment for the evolution of the budding yeast, Saccharomyces cerevisiae. To understand how the selection pressure shaped the yeast genome and gene regulation, we determined the genome sequence and transcriptome of a S. cerevisiae strain YHJ7 isolated from Chinese rice wine (Huangjiu), a popular traditional alcoholic beverage in China. By comparing the genome of YHJ7 to the lab strain S288c, a Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1, we identified many genomic sequence and structural variations in YHJ7, which are mainly located in subtelomeric regions, suggesting that these regions play an important role in genomic evolution between strains. In addition, our comparative transcriptome analysis between YHJ7 and S288c revealed a set of differentially expressed genes, including those involved in glucose transport (e.g., HXT2, HXT7) and oxidoredutase activity (e.g., AAD10, ADH7). Interestingly, many of these genomic and transcriptional variations are directly or indirectly associated with the adaptation of YHJ7 strain to its specific niches. Our molecular evolution analysis suggested that Japanese sake strains (K7/UC5) were derived from Chinese rice wine strains (YHJ7) at least approximately 2,300 years ago, providing the first molecular evidence elucidating the origin of Japanese sake strains. Our results depict interesting insights regarding the evolution of yeast during rice wine fermentation, and provided a valuable resource for genetic engineering to improve industrial wine-making strains. PMID:25212861

  18. Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose.

    PubMed

    Liu, Jing-Jing; Zhang, Guo-Chang; Oh, Eun Joong; Pathanibul, Panchalee; Turner, Timothy L; Jin, Yong-Su

    2016-09-20

    Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering. PMID:27457698

  19. A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82

    SciTech Connect

    Szent-Gyorgyi, C.

    1995-12-01

    This report seeks to characterize the activation of meiotic gene in terms of cis-acting DNA elements and their associated factors in Saccharomyces cerevisiae. It was found that vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element with a nearby bipartite repression element. The experiments described explore how two different regulatory pathways induce transcription by stimulating a single classical activation element, a nonspecific heat shock element. 81 refs., 10 figs., 1 tab.

  20. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    PubMed

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141

  1. Human G protein-coupled receptor studies in Saccharomyces cerevisiae.

    PubMed

    Liu, Rongfang; Wong, Winsy; IJzerman, Adriaan P

    2016-08-15

    G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30-40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a "null" background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems. PMID:26920251

  2. Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

    PubMed Central

    Overkamp, Karin M.; Bakker, Barbara M.; Kötter, Peter; Luttik, Marijke A. H.; van Dijken, Johannes P.; Pronk, Jack T.

    2002-01-01

    Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h−1 at 100 g of glucose · liter−1). In aerated batch cultures grown on 400 g of glucose · liter−1, this engineered S. cerevisiae strain produced over 200 g of glycerol · liter−1, corresponding to a molar yield of glycerol on glucose close to unity. PMID:12039737

  3. Exploring the Saccharomyces cerevisiae Volatile Metabolome: Indigenous versus Commercial Strains

    PubMed Central

    Alves, Zélia; Melo, André; Figueiredo, Ana Raquel; Coimbra, Manuel A.; Gomes, Ana C.; Rocha, Sílvia M.

    2015-01-01

    Winemaking is a highly industrialized process and a number of commercial Saccharomyces cerevisiae strains are used around the world, neglecting the diversity of native yeast strains that are responsible for the production of wines peculiar flavours. The aim of this study was to in-depth establish the S. cerevisiae volatile metabolome and to assess inter-strains variability. To fulfill this objective, two indigenous strains (BT2652 and BT2453 isolated from spontaneous fermentation of grapes collected in Bairrada Appellation, Portugal) and two commercial strains (CSc1 and CSc2) S. cerevisiae were analysed using a methodology based on advanced multidimensional gas chromatography (HS-SPME/GC×GC-ToFMS) tandem with multivariate analysis. A total of 257 volatile metabolites were identified, distributed over the chemical families of acetals, acids, alcohols, aldehydes, ketones, terpenic compounds, esters, ethers, furan-type compounds, hydrocarbons, pyrans, pyrazines and S-compounds. Some of these families are related with metabolic pathways of amino acid, carbohydrate and fatty acid metabolism as well as mono and sesquiterpenic biosynthesis. Principal Component Analysis (PCA) was used with a dataset comprising all variables (257 volatile components), and a distinction was observed between commercial and indigenous strains, which suggests inter-strains variability. In a second step, a subset containing esters and terpenic compounds (C10 and C15), metabolites of particular relevance to wine aroma, was also analysed using PCA. The terpenic and ester profiles express the strains variability and their potential contribution to the wine aromas, specially the BT2453, which produced the higher terpenic content. This research contributes to understand the metabolic diversity of indigenous wine microflora versus commercial strains and achieved knowledge that may be further exploited to produce wines with peculiar aroma properties. PMID:26600152

  4. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  5. Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

    PubMed Central

    2013-01-01

    Background Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l-1 ethanol (0.58 g l-1 h-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l-1 h-1). Conclusions In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases. PMID:24286270

  6. RNAi-assisted genome evolution in Saccharomyces cerevisiae for complex phenotype engineering.

    PubMed

    Si, Tong; Luo, Yunzi; Bao, Zehua; Zhao, Huimin

    2015-03-20

    A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution. PMID:24758359

  7. Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

    PubMed

    Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

    2014-10-01

    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. PMID:24318365

  8. Events associated with restoration by zinc of meiosis in apomictic Saccharomyces cerevisiae.

    PubMed Central

    Bilinski, C A; Miller, J J; Girvitz, S C

    1983-01-01

    The effects of nutritional alterations (carbon source and zinc) on nuclear division and protein synthesis during apomictic and meiotic development in Saccharomyces cerevisiae 19e1 were investigated. Unlike cells cultivated under meiosis-promoting conditions, cells cultured under apomixis-promoting conditions exhibited extensive protein synthesis during the first 3 h of incubation in sporulation medium, and nuclear divisions were evident during this time. Cycloheximide treatment of the latter cells induced meiosis, and maximum yields of meiotic asci resulted when this treatment was given for the first 3 h in sporulation medium. The results indicate that the decision concerning which developmental route cells will follow is made shortly after transfer to sporulation medium. Electrophoretic analysis of labeled proteins synthesized during sporulation revealed bands unique to both developmental routes. Images PMID:6350265

  9. Effects of dietary antibiotic growth promoter and Saccharomyces cerevisiae fermentation product on production, intestinal bacterial community, and nonspecific immunity of hybrid tilapia (Oreochromis niloticus female x Oreochromis aureus male).

    PubMed

    He, S; Zhou, Z; Meng, K; Zhao, H; Yao, B; Ringø, E; Yoon, I

    2011-01-01

    To investigate the effects of a dietary antibiotic growth promoter (florfenicol) and a Saccharomyces cerevisiae fermentation product (DVAQUA) on growth, G:F, daily feed intake, intestinal bacterial community, and nonspecific immunity of hybrid tilapia (Oreochromis niloticus ♀ × Oreochromis aureus ♂), a 16-wk feeding trial was conducted in a recirculating aquaculture system. Four feeding regimens were evaluated: control, dietary florenicol (0.02 g/kg; 16 wk), dietary DVAQUA (0.5 g/kg; 16 wk), and sequential use of florenicol (0.02 g/kg; 8 wk), and DVAQUA (0.5 g/kg; 8 wk). Each regimen had 4 replicate tanks (0.5 × 0.5 × 0.5 m) and each tank contained 12 fish (initial BW: 46.88 ± 0.38 g). Dietary florfenicol improved growth (P = 0.089), G:F (P = 0.036), and serum complement component concentrations (P < 0.001) of hybrid tilapia. However, the compound decreased the estimated intestinal bacterial count estimated by rpoB quantitative PCR (P < 0.001) and bacterial diversity (visual band numbers, Shannon diversity index, and Shannon equitability index based on 16S rDNA V3 denaturing gradient gel electrophoresis fingerprints) compared with the control. Although sequential use of florfenicol and DVAQUA improved growth and G:F numerically to a similar extent as dietary florfenicol, and increased intestinal bacterial count to normal quantities, the sequential use of florenicol and DVAQUA decreased intestinal bacterial diversity (visual band numbers, Shannon diversity index, and Shannon equitability index) as well as serum complement component concentrations (P < 0.001) compared with their respective use and the control. These findings might be negatively related to disease control and host defense, and the sequential use of florenicol and DVAQUA should be practiced with caution. Feeding DAVQUA to the fish improved nonspecific immunity and increased intestinal bacterial count and bacterial diversity, but further research, including challenge studies, should be conducted

  10. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    PubMed Central

    Pablo-Hernando, M Evangelina; Arnaiz-Pita, Yolanda; Tachikawa, Hiroyuki; del Rey, Francisco; Neiman, Aaron M; Vázquez de Aldana, Carlos R

    2008-01-01

    Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources. PMID:18826657

  11. Engineering the monomer composition of polyhydroxyalkanoates synthesized in Saccharomyces cerevisiae.

    PubMed

    Zhang, Bo; Carlson, Ross; Srienc, Friedrich

    2006-01-01

    Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical beta-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C6 to C14) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as beta-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties. PMID:16391089

  12. Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.

    PubMed Central

    Zitomer, R S; Walthall, D A; Rymond, B C; Hollenberg, C P

    1984-01-01

    A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results. PMID:6390186

  13. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-06-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  14. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed Central

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  15. Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae.

    PubMed

    Vuralhan, Zeynep; Morais, Marcos A; Tai, Siew-Leng; Piper, Matthew D W; Pronk, Jack T

    2003-08-01

    Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding alpha-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The K(m) and V(max) values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering

  16. Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

    PubMed Central

    Perry, J R; Basrai, M A; Steiner, H Y; Naider, F; Becker, J M

    1994-01-01

    We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene. Images PMID:8264579

  17. Effects of microcystin-LR on Saccharomyces cerevisiae growth, oxidative stress and apoptosis.

    PubMed

    Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, Vitor

    2014-11-01

    Microcystins (MC) are cyanotoxins occurring globally, known for causing acute hepatotoxicity in humans/animals, tumor promotion in animals and potential carcinogenicity. The mechanism of MC toxicity is considered a multi-pathway process involving the inhibition of protein phosphatases PP1/PP2A and the production of reactive oxygen species (ROS). However, their mechanism of action is not fully characterized, thus hampering the complete hazard identification. In this study, we evaluated the effect of several microcystin-LR concentrations on the growth, ROS levels, antioxidant system response and apoptosis induction on Saccharomyces cerevisiae. Our results showed that the growth of S. cerevisiae was not inhibited when compared to control cells. However, the staining of cells with DHR123 and DHE revealed an intracellular increase of the ROS levels. This ROS increase resulted in an augment of catalase activity and inhibition of SOD. All these facts suggest that hydrogen peroxide was the main ROS induced by MCLR. Signs of apoptosis were also detected in the cells exposed to toxin. Our results show that S. cerevisiae VL3 displays MCLR toxicity effects known to occur in higher eukaryotes and confirmed that it can be a simple and good model to help further in the elucidation of MCLR molecular mechanisms of toxicity. PMID:25157802

  18. Characterization of Human RNA Polymerase III Identifies Orthologues for Saccharomyces cerevisiae RNA Polymerase III Subunits

    PubMed Central

    Hu, Ping; Wu, Si; Sun, Yuling; Yuan, Chih-Chi; Kobayashi, Ryuji; Myers, Michael P.; Hernandez, Nouria

    2002-01-01

    Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription. PMID:12391170

  19. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress

    PubMed Central

    Nguyen, Trinh T. M.; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin. PMID:26696995

  20. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

    PubMed

    Nguyen, Trinh T M; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin. PMID:26696995

  1. Higher-Order Septin Assembly Is Driven by GTP-Promoted Conformational Changes: Evidence From Unbiased Mutational Analysis in Saccharomyces cerevisiae

    PubMed Central

    Weems, Andrew D.; Johnson, Courtney R.; Argueso, Juan Lucas; McMurray, Michael A.

    2014-01-01

    Septin proteins bind GTP and heterooligomerize into filaments with conserved functions across a wide range of eukaryotes. Most septins hydrolyze GTP, altering the oligomerization interfaces; yet mutations designed to abolish nucleotide binding or hydrolysis by yeast septins perturb function only at high temperatures. Here, we apply an unbiased mutational approach to this problem. Mutations causing defects at high temperature mapped exclusively to the oligomerization interface encompassing the GTP-binding pocket, or to the pocket itself. Strikingly, cold-sensitive defects arise when certain of these same mutations are coexpressed with a wild-type allele, suggestive of a novel mode of dominance involving incompatibility between mutant and wild-type molecules at the septin–septin interfaces that mediate filament polymerization. A different cold-sensitive mutant harbors a substitution in an unstudied but highly conserved region of the septin Cdc12. A homologous domain in the small GTPase Ran allosterically regulates GTP-binding domain conformations, pointing to a possible new functional domain in some septins. Finally, we identify a mutation in septin Cdc3 that restores the high-temperature assembly competence of a mutant allele of septin Cdc10, likely by adopting a conformation more compatible with nucleotide-free Cdc10. Taken together, our findings demonstrate that GTP binding and hydrolysis promote, but are not required for, one-time events—presumably oligomerization-associated conformational changes—during assembly of the building blocks of septin filaments. Restrictive temperatures impose conformational constraints on mutant septin proteins, preventing new assembly and in certain cases destabilizing existing assemblies. These insights from yeast relate directly to disease-causing mutations in human septins. PMID:24398420

  2. The Spo12 protein of Saccharomyces cerevisiae: a regulator of mitotic exit whose cell cycle-dependent degradation is mediated by the anaphase-promoting complex.

    PubMed Central

    Shah, R; Jensen, S; Frenz, L M; Johnson, A L; Johnston, L H

    2001-01-01

    The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis. PMID:11729145

  3. β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media.

    PubMed

    Lange, Nicole; Steinbüchel, Alexander

    2011-09-01

    A recombinant Saccharomyces cerevisiae strain was used for the production of β-carotene. The episomal plasmid YEplac195YB/I/E was extended by a gene coding for the mevalonate kinase (mvaK1) from Staphylococcus aureus. The adh1 promoter was chosen for constitutive expression of mvaK1. The recombinant strain S. cerevisiae G175 (YEplac-CaroSA) synthesised β-carotene by expressing the carotenogenic genes of Xanthophyllomyces dendrorhous together with the mvaK1 gene. Cells of this strain were investigated for their carotenoid contents in YNB and YPD media. A corresponding mvaK1 transcript in the recombinant yeast host was verified. Growth experiments of a specific erg12 deletion mutant showed that the mevalonate kinase (MvaK1) was able to complement the function of the deleted native mevalonate kinase (Erg12) from S. cerevisiae in the MVA pathway under control of the constitutive adh1 promoter. Cells of S. cerevisiae G175 (YEplac-CaroSA) exhibited high plasmid stability under either selective or non-selective cultivation conditions. Time course experiments demonstrated high plasmid stability even over extended cultivation periods. Carotenoid production was therefore also stable in larger culture volumes. Due to the stability of the plasmid, cultivation of the cells in complex YPD medium was possible, and 14.3 mg β-carotene per litre and a cell density of 9 g cell dry matter (CDM) per litre were achieved. The highest amount of 3,897 μg β-carotene per gramme CDM at a cell density of 1 g CDM per litre was measured after cultivation of the cells in YNB medium with glucose as sole carbon source. PMID:21573686

  4. Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

    PubMed Central

    Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.

    2013-01-01

    Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment. PMID:23614011

  5. Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

    NASA Technical Reports Server (NTRS)

    Johanson, Kelly; Allen, Patricia L.; Lewis, Fawn; Cubano, Luis A.; Hyman, Linda E.; Hammond, Timothy G.

    2002-01-01

    This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

  6. Effect of citrus pulp on the viability of the probiotic Saccharomyces cerevisiae bouldarii (Levucell) and subsequent effects in presence of pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The probiotic Saccharomyces cerevisiae subtype boulardii (i.e., Levucell) is commonly provided to weaned and nursing sows to promote intestinal health through limiting the colonization of pathogens. Recent research from our group identified that supplementation of this probiotic to feed containing t...

  7. Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

    PubMed Central

    Swedes, J S; Dial, M E; McLaughlin, C S

    1979-01-01

    Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. PMID:374362

  8. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae

    PubMed Central

    Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M

    2014-01-01

    The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth. PMID:24483210

  9. Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae

    SciTech Connect

    Wong, H.; Wilke, C.R.; Blanch, H.W.

    1983-05-01

    Using Saccharomyces cerevisiae as a model system, a basic study of the nutritional and environmental factors in ethanol fermentation was carried out to provide fundamental and practical bases for design of fermentation media and culture conditions. The requirements for all active medium components need to be determined in order to establish balanced media, which are important to reduce raw materials costs and to minimize inhibition from buildup of excess feed components in recycle processes with selective ethanol removal. Pulse injection of nutrients into continuous cultures was an effective method for screening active nutrients. In a systematic sensitivity analysis the effect of feed concentration of these individual nutrients was then determined and allowed formulation of media optimal with respect to the major fermentation parameters. Biotin, pantothenate, myo-inositol, potassium and phosphates appeared to stimulate growth preferentially to ethanol production. In contrast, thiamine and pyridoxine appeared to enhance specific ethanol productivity. The effect of ammonium sulfate depended on concentration. A conceptual model was proposed to relate the effects of these nutrients to biochemical pathways and functions. With these data and model the minimum cost combination of raw materials to achieve a medium of well defined components can be determined with a linear program. This computer program shows that many growth factors and minerals can be added to media more economically as pure components than as fractions of complex factors. 225 references, 61 figures, 54 tables.

  10. Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae

    SciTech Connect

    Wong, H.

    1983-01-01

    Using Saccharomyces cerevisiae as a model system, a basic study of the nutritional and environmental factors in ethanol fermentation was carried out to provide fundamental and practical bases for design of fermentation media and culture conditions. The requirements for all active medium components need to be determined in order to establish balanced media, which are important to reduce raw materials costs and to minimize inhibition from build-up of excess feed components in recycle processes with selective ethanol removal. The effect of feed concentration of individual nutrients was determined and allowed formulation of media optimal with respect to the major fermentation parameters. Biotin, pantothenate, myoinositol, potassium, and phosphates appeared to stimulate growth preferentially to ethanol production. Thiamine and pyridoxine appeared to have the opposite effect. A conceptual model was proposed to relate the effects of these nutrients to biochemical pathways and functions. The minimum cost combination of raw materials to achieve a medium of well defined components can be determined with a linear program. The effect of dissolved oxygen was studied from essentially zero to 346 mm Hg oxygen tension, showing a continuous decline in specific ethanol productivity with increasing oxygen over this range. Long term continuous cultures resulted in decreased media requirements for growth factors and increased tolerance for ethanol inhibition, most probably through adaptation. An ethanol productivity of 5.6 g/l-hr in continuous culture was achieved with a completely synthetic medium with the improved culture.

  11. Transcriptional Response of Saccharomyces cerevisiae to Desiccation and Rehydration†

    PubMed Central

    Singh, Jatinder; Kumar, Deept; Ramakrishnan, Naren; Singhal, Vibha; Jervis, Jody; Garst, James F.; Slaughter, Stephen M.; DeSantis, Andrea M.; Potts, Malcolm; Helm, Richard F.

    2005-01-01

    A transcriptional analysis of the response of Saccharomyces cerevisiae strain BY4743 to controlled air-drying (desiccation) and subsequent rehydration under minimal glucose conditions was performed. Expression of genes involved in fatty acid oxidation and the glyoxylate cycle was observed to increase during drying and remained in this state during the rehydration phase. When the BY4743 expression profile for the dried sample was compared to that of a commercially prepared dry active yeast, strikingly similar expression changes were observed. The fact that these two samples, dried by different means, possessed very similar transcriptional profiles supports the hypothesis that the response to desiccation is a coordinated event independent of the particular conditions involved in water removal. Similarities between “stationary-phase-essential genes” and those upregulated during desiccation were also noted, suggesting commonalities in different routes to reduced metabolic states. Trends in extracellular and intracellular glucose and trehalose levels suggested that the cells were in a “holding pattern” during the rehydration phase, a concept that was reinforced by cell cycle analyses. Application of a “redescription mining” algorithm suggested that sulfur metabolism is important for cell survival during desiccation and rehydration. PMID:16332871

  12. Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress

    PubMed Central

    Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping

    2016-01-01

    Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154

  13. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    SciTech Connect

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offs between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.

  14. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    DOE PAGESBeta

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offsmore » between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.« less

  15. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  16. Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production.

    PubMed

    Feng, Xueyang; Lian, Jiazhang; Zhao, Huimin

    2015-01-01

    Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45mg/L to 71mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71mg/L to 140mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140mg/L to 330mg/L). Through fed-batch fermentation using resting cells, over 1.1g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date. PMID:25466225

  17. MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Gustin, Michael C.; Albertyn, Jacobus; Alexander, Matthew; Davenport, Kenneth

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area. PMID:9841672

  18. Water-Transfer Slows Aging in Saccharomyces cerevisiae

    PubMed Central

    Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana

    2016-01-01

    Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process. PMID:26862897

  19. A novel selection system for chromosome translocations in Saccharomyces cerevisiae.

    PubMed Central

    Tennyson, Rachel B; Ebran, Nathalie; Herrera, Anissa E; Lindsley, Janet E

    2002-01-01

    Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations. PMID:11973293

  20. Water-Transfer Slows Aging in Saccharomyces cerevisiae.

    PubMed

    Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana

    2016-01-01

    Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process. PMID:26862897

  1. Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae.

    PubMed Central

    Thielen, J; Ciriacy, M

    1991-01-01

    As reported previously, Saccharomyces cerevisiae cells deficient in all four known genes coding for alcohol dehydrogenases (ADH1 through ADH4) produce considerable amounts of ethanol during aerobic growth on glucose. It has been suggested that ethanol production in such adh0 cells is a corollary of acetaldehyde dismutation in mitochondria. This could be substantiated further by showing that mitochondrial ethanol formation requires functional electron transport, while the proton gradient or oxidative phosphorylation does not interfere with reduction of acetaldehyde in isolated mitochondria. This acetaldehyde-reducing activity is different from classical alcohol dehydrogenases in that it is associated with the inner mitochondrial membrane and also is unable to carry out ethanol oxidation. The putative cofactor is NADH + H+ generated by a soluble, matrix-located aldehyde dehydrogenase upon acetaldehyde oxidation to acetate. This enzyme has been purified from mitochondria of glucose-grown cells. It is clearly different from the known mitochondrial aldehyde dehydrogenase, which is absent in glucose-grown cells. Both acetaldehyde-reducing and acetaldehyde-oxidizing activities are also present in the mitochondrial fraction of fermentation-proficient (ADH+) cells. Mitochondrial acetaldehyde dismutation may have some significance in the removal of surplus acetaldehyde and in the formation of acetate in mitochondria during aerobic glucose fermentation. Images FIG. 4 PMID:1938903

  2. Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae.

    PubMed

    Ganeva, Valentina; Stefanova, Debora; Angelova, Boyana; Galutzov, Bojidar; Velasco, Isabel; Arévalo-Rodríguez, Miguel

    2015-10-10

    Yeasts are one of the most commonly used systems for recombinant protein production. When the protein is intracelullarly expressed the first step comprises a cell lysis, achieved usually by a mechanical disintegration. This leads to non-selective liberation of the cytoplasmic content, which complicates the following downstream process. Here, we present a new approach suitable for more selective and efficient recovery of large intracellular proteins from yeast, based on the combination of electropermeabilisation and subsequent treatment with lytic enzyme. The experiments were performed with Saccharomyces cerevisiae strains expressing LYTAG-β-galactosidase from Escherichia coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25ms duration and field intensity of 4.3-5.4kV/cm. In the presence of a reducing agent the cells released approximately 80% of the total protein 4h after electrical treatment. At the same conditions the release of the recombinant protein was very slow, reaching 45% from total activity 20h after pulse application. The great difference in the release kinetics enabled to remove a part of the total protein, without significant loss of β-galactosidase activity, only by substituting the incubation buffer. The subsequent addition of lyticase (1-2U/ml) led to recovery of approximately 70% from the recombinant enzyme, with a factor of purification 2.6, without provoking a significant cell lysis. The applicability of similar protocol for liberation of large recombinant and native proteins from yeast is discussed. PMID:26142064

  3. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

    The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

  4. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  5. Systematic Identification of Balanced Transposition Polymorphisms in Saccharomyces cerevisiae

    PubMed Central

    Faddah, Dina A.; Ganko, Eric W.; McCoach, Caroline; Pickrell, Joseph K.; Hanlon, Sean E.; Mann, Frederick G.; Mieczkowska, Joanna O.; Jones, Corbin D.; Lieb, Jason D.; Vision, Todd J.

    2009-01-01

    High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH) to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90) and a polymorphic wild isolate (Y101) through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism. PMID:19503594

  6. Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations.

    PubMed

    Sarais, I; Manzano, M; De Bertoldi, M; Romandini, P; Beltramini, M; Salvato, B; Rocco, G P

    1994-07-01

    A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mM). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent. PMID:8043987

  7. Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

    PubMed

    Buchanan, Bryce W; Lloyd, Michael E; Engle, Sarah M; Rubenstein, Eric M

    2016-01-01

    Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation. PMID:27167179

  8. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  9. Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae.

    PubMed

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2014-10-01

    Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans. PMID:24970458

  10. mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

    PubMed Central

    Jerome, J F; Jaehning, J A

    1986-01-01

    We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708