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Sample records for arming yeast expressing

  1. Shuffling Yeast Gene Expression Data

    OpenAIRE

    Bilke, Sven

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent ...

  2. Shuffling Yeast Gene Expression Data

    CERN Document Server

    Bilke, S

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent cell clock is identified. The capability of the algorithm to extract information about signal flow in the regulatory network underlying the expression patterns is demonstrated.

  3. A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

    Directory of Open Access Journals (Sweden)

    Zhang Tingting

    2012-12-01

    Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

  4. Super-paramagnetic clustering of yeast gene expression profiles

    CERN Document Server

    Getz, G; Domany, E; Zhang, M Q

    2000-01-01

    High-density DNA arrays, used to monitor gene expression at a genomic scale, have produced vast amounts of information which require the development of efficient computational methods to analyze them. The important first step is to extract the fundamental patterns of gene expression inherent in the data. This paper describes the application of a novel clustering algorithm, Super-Paramagnetic Clustering (SPC) to analysis of gene expression profiles that were generated recently during a study of the yeast cell cycle. SPC was used to organize genes into biologically relevant clusters that are suggestive for their co-regulation. Some of the advantages of SPC are its robustness against noise and initialization, a clear signature of cluster formation and splitting, and an unsupervised self-organized determination of the number of clusters at each resolution. Our analysis revealed interesting correlated behavior of several groups of genes which has not been previously identified.

  5. A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

    OpenAIRE

    Zhang Tingting; Sun Lin; Xin Ying; Ma Lixia; Zhang Youyou; Wang Xin; Xu Kun; Ren Chonghua; Zhang Cunfang; Chen Zhilong; Yang Hanjiang; Zhang Zhiying

    2012-01-01

    Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concern...

  6. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    Science.gov (United States)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  7. Inositol and Phosphatidylinositol Mediated Glucose Derepression, Gene Expression and Invertase Secretion in Yeasts

    Institute of Scientific and Technical Information of China (English)

    Zhen-Ming CHI; Jun-Feng LI; Xiang-Hong WANG; Shu-Min YAO

    2004-01-01

    Glucose repression occurs in many yeast species and some filamentous fungi, and it represses the expression and secretion of many intracellular and extracellular proteins. In recent years, it has been found that many biochemical reactions in yeast cells are mediated by phosphatidylinositol (PI)-type signaling pathway. However, little is known about the relationships between PI-type signaling and glucose repression,gene expression and invertase secretion in yeasts. Many evidences in our previous studies showed that glucose repression, invertase secretion, gene expression and cell growth were mediated by inositol and PI in Saccharomyces and Schizosaccharomyces. The elucidation of the new regulatory mechanisms of protein secretion, gene expression and glucose repression would be an entirely new aspect of inositol and PI-type signaling regulation in yeasts.

  8. Isolated yeast promoter sequence and a method of regulated heterologous expression

    Science.gov (United States)

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

    2005-05-31

    The present invention provides the promoter clone discovery of a glucoamylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated glucoamylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  9. Yeast Endoplasmic Reticulum Sequestration Screening for the Engineering of Proteases from Libraries Expressed in Yeast.

    Science.gov (United States)

    Yi, Li; Taft, Joseph M; Li, Qing; Gebhard, Mark C; Georgiou, George; Iverson, Brent L

    2015-01-01

    There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis. The method described here allows facile screening of large libraries, and of either protease or substrate variants, including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition, the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease, and the human Abelson tyrosine kinase. PMID:26060071

  10. Yeast PPR proteins, watchdogs of mitochondrial gene expression

    OpenAIRE

    Herbert, Christopher J.; Golik, Pawel; Bonnefoy, Nathalie

    2013-01-01

    PPR proteins are a family of ubiquitous RNA-binding factors, found in all the Eukaryotic lineages, and are particularly numerous in higher plants. According to recent bioinformatic analyses, yeast genomes encode from 10 (in S. pombe) to 15 (in S. cerevisiae) PPR proteins. All of these proteins are mitochondrial and very often interact with the mitochondrial membrane. Apart from the general factors, RNA polymerase and RNase P, most yeast PPR proteins are involved in the stability and/or transl...

  11. Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Moussa Manal

    2012-12-01

    Full Text Available Abstract Background Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA and streptokinase (SK. Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK, or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. Results The development of an efficient large scale (80 L Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.. Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth of biologically active rSAK-2 of expected molecular weight was achieved. Conclusion The optimized production process

  12. The identification and high level expression of a protein encoded by the yeast Ty element.

    OpenAIRE

    Dobson, M J; Mellor, J; Fulton, A.M.; Roberts, N A; Bowen, B A; Kingsman, S M; Kingsman, A J

    1984-01-01

    Transcription of the yeast Ty element, Ty1 -15, was placed under the control of the efficient PGK promoter on the yeast expression vector, pMA91 . In extracts of yeast transformants containing these constructions a new 52 K basic polypeptide was detected by one- and two-dimensional electrophoresis and was shown, by hybrid arrested translation, to be specifically encoded by the Ty element. The protein coding region was mapped to the first 1.45 kb of the transcribed region of Ty1 -15. These dat...

  13. Nitrogen starvation induces expression of Lg-FLO1 and flocculation in bottom-fermenting yeast.

    Science.gov (United States)

    Ogata, Tomoo

    2012-11-01

    When exponentially growing cells of bottom-fermenting yeast were starved for nitrogen or were grown on proline (a non-preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg-FLO1, which is required for flocculation of bottom-fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom-fermenting yeast is under the control of a nitrogen catabolite repression (NCR)-like mechanism.

  14. Yeast expression proteomics by high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Walther, Tobias C; Olsen, Jesper Velgaard; Mann, Matthias

    2010-01-01

    Comprehensive analysis of yeast as a model system requires to reliably determine its composition. Systematic approaches to globally determine the abundance of RNAs have existed for more than a decade and measurements of mRNAs are widely used as proxies for detecting changes in protein abundance. ...

  15. In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.

    OpenAIRE

    Deforce, L; Tomizawa, K; Ito, N; Farrens, D; Song, P S; Furuya, M.

    1991-01-01

    Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids fr...

  16. Functional expression of parasite drug targets and their human orthologs in yeast.

    Directory of Open Access Journals (Sweden)

    Elizabeth Bilsland

    2011-10-01

    Full Text Available BACKGROUND: The exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents. METHODOLOGY/PRINCIPAL FINDINGS: Using pyrimethamine/dihydrofolate reductase (DHFR as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p expressing yeast ((ScDFR1, human ((HsDHFR, Schistosoma ((SmDHFR, and Trypanosoma ((TbDHFR and (TcDHFR DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium ((PfDHFR and (PvDHFR DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR ((Pfdhfr(51I,59R,108N are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs and N-myristoyl transferases (NMTs. CONCLUSIONS/SIGNIFICANCE: We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents.

  17. Analysis of a 26,756 bp segment from the left arm of yeast chromosome IV.

    Science.gov (United States)

    Wölfl, S; Hanemann, V; Saluz, H P

    1996-12-01

    The nucleotide sequence of a 26.7 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome IV is presented. An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene. These ORFs include the genes encoding the large subunit of RNA polymerase II, the biotin apo-protein ligase, an ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor, and the sequence encoding the protein phosphatase 2A. Further sequence analysis revealed a short ORF encoding the ribosomal protein YL41B, an intron in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome. The potential biological relevance of these findings is discussed. PMID:8972577

  18. Analysis of heterogeneity of gene products(interferon)expressed in yeast

    Institute of Scientific and Technical Information of China (English)

    王洪海; 高卜渝; 陈佩丽; 董灵; 李育阳

    1995-01-01

    FPLC,SDS-PAGE and Western blot techniques are used to analyse the heterogeneity ofinterferon αA(IFN-αA) expressed in yeast.The heterogeneity consists of (i) the presence of IFN polymer,(ii)partial processing of signal leader peptide and (iii) internal degradation.The reasons for heterogeneity ofgene products in expression system of yeast are analysed.The methods of avoiding heterogeneity,such asdepolymerization,adding inhibitors of protease to the culture supernatant,the oligonucleotide-directed deletionmutagenesis and improvements of fermentation,are discussed.

  19. Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display.

    Science.gov (United States)

    Parthasarathy, Ranganath; Subramanian, Shyamsundar; Boder, Eric T; Discher, Dennis E

    2006-01-01

    Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.

  20. Expression of a Ripening-Related Avocado (Persea americana) Cytochrome P450 in Yeast.

    Science.gov (United States)

    Bozak, K R; O'keefe, D P; Christoffersen, R E

    1992-12-01

    One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex.

  1. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  2. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    Science.gov (United States)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  3. Use of CYP52A2A promoter to increase gene expression in yeast

    Science.gov (United States)

    Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

    2004-01-06

    A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

  4. Brewing yeast genomes and genome-wide expression and proteome profiling during fermentation.

    Science.gov (United States)

    Smart, Katherine A

    2007-11-01

    The genome structure, ancestry and instability of the brewing yeast strains have received considerable attention. The hybrid nature of brewing lager yeast strains provides adaptive potential but yields genome instability which can adversely affect fermentation performance. The requirement to differentiate between production strains and assess master cultures for genomic instability has led to significant adoption of specialized molecular tool kits by the industry. Furthermore, the development of genome-wide transcriptional and protein expression technologies has generated significant interest from brewers. The opportunity presented to explore, and the concurrent requirement to understand both, the constraints and potential of their strains to generate existing and new products during fermentation is discussed.

  5. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach;

    , (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression...

  6. Palytoxin induces K+ efflux from yeast cells expressing the mammalian sodium pump.

    Science.gov (United States)

    Scheiner-Bobis, G; Meyer zu Heringdorf, D; Christ, M; Habermann, E

    1994-06-01

    Palytoxin causes potassium efflux and sodium influx in all investigated animals cells. Much evidence points to the sodium pump (Na+/K(+)-ATPase) as the target of the toxin. A heterologous expression system for mammalian Na+/K(+)-ATPase in the brewers yeast Saccharomyces cerevisiae has been used to test this hypothesis. Yeast cells do not contain endogenous sodium pumps but can be transformed with vectors coding for the alpha and beta subunits of the mammalian sodium pump. We now show that transformed yeast cells expressing both alpha and beta subunits of Na+/K(+)-ATPase are highly sensitive to the toxin, as measured by the loss of intracellular potassium. Palytoxin-induced potassium efflux is completely inhibited by 500 microM ouabain. In contrast, nontransformed yeast cells or cells expressing either the alpha or beta subunits are insensitive to palytoxin. Thus, the alpha/beta heterodimer of the sodium pump is required for the release of potassium induced by palytoxin. The results suggest that palytoxin converts the sodium pump into an open channel, allowing the passage of alkali ions.

  7. Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

    Science.gov (United States)

    Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal

    2015-12-01

    Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. PMID:26555900

  8. Analysis of the structure and function of EMRE in a yeast expression system.

    Science.gov (United States)

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore. PMID:27001609

  9. Analysis of the structure and function of EMRE in a yeast expression system.

    Science.gov (United States)

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.

  10. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.

  11. Evidence for widespread adaptive evolution of gene expression in budding yeast.

    Science.gov (United States)

    Fraser, Hunter B; Moses, Alan M; Schadt, Eric E

    2010-02-16

    Changes in gene expression have been proposed to underlie many, or even most, adaptive differences between species. Despite the increasing acceptance of this view, only a handful of cases of adaptive gene expression evolution have been demonstrated. To address this discrepancy, we introduce a simple test for lineage-specific selection on gene expression. Applying the test to genome-wide gene expression data from the budding yeast Saccharomyces cerevisiae, we find that hundreds of gene expression levels have been subject to lineage-specific selection. Comparing these findings with independent population genetic evidence of selective sweeps suggests that this lineage-specific selection has resulted in recent sweeps at over a hundred genes, most of which led to increased transcript levels. Examination of the implicated genes revealed a specific biochemical pathway--ergosterol biosynthesis--where the expression of multiple genes has been subject to selection for reduced levels. In sum, these results suggest that adaptive evolution of gene expression is common in yeast, that regulatory adaptation can occur at the level of entire pathways, and that similar genome-wide scans may be possible in other species, including humans.

  12. Expression of a bacterial ice nucleation gene in a yeast Saccharomyces cerevisiae and its possible application in food freezing processes.

    Science.gov (United States)

    Hwang, W Z; Coetzer, C; Tumer, N E; Lee, T C

    2001-10-01

    A 3.6 kb ice nucleation gene (ina) isolated from Erwinia herbicola was placed under control of the galactose-inducible promoter (GAL1) and introduced into Saccharomyces cerevisiae. Yeast transformants showed increased ice nucleation activity over untransformed controls. The freezing temperature of a small volume of water droplets containing yeast cells was increased from approximately -13 degrees C in the untransformed controls to -6 degrees C in ina-expressing (Ina(+)) transformants. Lower temperature growth of Ina(+) yeast at temperatures below 25 degrees C was required for the expression of ice nucleation activity. Shift of temperature to 5-20 degrees C could induce the ice nucleation activity of Ina(+) yeast when grown at 25 degrees C, and maximum ice nucleation activity was achieved after induction at 5 degrees C for approximately 12 h. The effects of Ina(+) yeast on freezing and texturization of several food materials was also demonstrated. PMID:11600004

  13. Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg.

    Science.gov (United States)

    Shin, Dong-Il; Song, Kyu-Seon; Park, Hee-Sung

    2015-03-01

    Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters. PMID:25374008

  14. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    Science.gov (United States)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  15. Yeast genomic expression patterns in response to low-shear modeled microgravity

    Directory of Open Access Journals (Sweden)

    Altenburg Sara D

    2007-01-01

    Full Text Available Abstract The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs, allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. Results LSMMG differentially changed the expression of a significant number of genes (1372 when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome

  16. Expression strategies of the yeast retrotransposon Ty: a short sequence directs ribosomal frameshifting.

    OpenAIRE

    Wilson, W; Malim, M H; Mellor, J; Kingsman, A J; Kingsman, S M

    1986-01-01

    The Ty element of yeast is a member of a class of eukaryotic transposons which bear a striking resemblance to retroviral proviruses in their structure and expression strategies (1,2). A direct comparison can be drawn between the production of a fusion protein encoded by Ty, resulting from a frameshift event which fuses two out-of-phase open reading frames TYA and TYB, and the production of Pr180gag-pol in a retrovirus such as Rous Sarcoma Virus (RSV) (3,4). We present data which shows, defini...

  17. [Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

    Science.gov (United States)

    Terekhov, S S; Bobik, T V; Mokrushina, Yu A; Stepanova, A V; Aleksandrova, N M; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2016-01-01

    A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day). PMID:27266247

  18. Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase.

    Science.gov (United States)

    Singh, Shailendra; Lee, Wonkyu; Dasilva, Nancy A; Mulchandani, Ashok; Chen, Wilfred

    2008-02-01

    Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

  19. Improvement of fermentation ability under baking-associated stress conditions by altering the POG1 gene expression in baker's yeast.

    Science.gov (United States)

    Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Oshiro, Satoshi; Shima, Jun; Takagi, Hiroshi

    2013-08-01

    During the bread-making process, yeast cells are exposed to many types of baking-associated stress. There is thus a demand within the baking industry for yeast strains with high fermentation abilities under these stress conditions. The POG1 gene, encoding a putative transcription factor involved in cell cycle regulation, is a multicopy suppressor of the yeast Saccharomyces cerevisiae E3 ubiquitin ligase Rsp5 mutant. The pog1 mutant is sensitive to various stresses. Our results suggested that the POG1 gene is involved in stress tolerance in yeast cells. In this study, we showed that overexpression of the POG1 gene in baker's yeast conferred increased fermentation ability in high-sucrose-containing dough, which is used for sweet dough baking. Furthermore, deletion of the POG1 gene drastically increased the fermentation ability in bread dough after freeze-thaw stress, which would be a useful characteristic for frozen dough baking. Thus, the engineering of yeast strains to control the POG1 gene expression level would be a novel method for molecular breeding of baker's yeast. PMID:23800735

  20. Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains.

    Science.gov (United States)

    Tanghe, An; Van Dijck, Patrick; Dumortier, Françoise; Teunissen, Aloys; Hohmann, Stefan; Thevelein, Johan M

    2002-12-01

    Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications. PMID:12450819

  1. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  2. Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

    Directory of Open Access Journals (Sweden)

    Daria Jacob

    Full Text Available Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV nucleoprotein (N known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs. Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen

  3. Inference of transcription modification in long-live yeast strains from their expression profiles

    Directory of Open Access Journals (Sweden)

    Longo Valter D

    2007-07-01

    Full Text Available Abstract Background Three kinases: Sch9, PKA and TOR, are suggested to be involved in both the replicative and chronological ageing in yeast. They function in pathways whose down-regulation leads to life span extension. Several stress response proteins, including two transcription factors Msn2 and Msn4, mediate the longevity extension phenotype associated with decreased activity of either Sch9, PKA, or TOR. However, the mechanisms of longevity, especially the underlying transcription program have not been fully understood. Results We measured the gene expression profiles in wild type yeast and three long-lived mutants: sch9Δ, ras2Δ, and tor1Δ. To elucidate the transcription program that may account for the longevity extension, we identified the transcription factors that are systematically and significantly associated with the expression differentiation in these mutants with respect to wild type by integrating microarray expression data with motif and ChIP-chip data, respectively. Our analysis suggests that three stress response transcription factors, Msn2, Msn4 and Gis1, are activated in all the three mutants. We also identify some other transcription factors such as Fhl1 and Hsf1, which may also be involved in the transcriptional modification in the long-lived mutants. Conclusion Combining microarray expression data with other data sources such as motif and ChIP-chip data provides biological insights into the transcription modification that leads to life span extension. In the chronologically long-lived mutant: sch9Δ, ras2Δ, and tor1Δ, several common stress response transcription factors are activated compared with the wild type according to our systematic transcription inference.

  4. Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

    Science.gov (United States)

    Kondrák, Mihály; Marincs, Ferenc; Kalapos, Balázs; Juhász, Zsófia; Bánfalvi, Zsófia

    2011-01-01

    Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

  5. Transcriptome analysis of potato leaves expressing the trehalose-6-phosphate synthase 1 gene of yeast.

    Directory of Open Access Journals (Sweden)

    Mihály Kondrák

    Full Text Available Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1 gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative protein-protein interactions involving some of the up- and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.

  6. [Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts Saccharomyces cerevisiae and Pichia pastoris].

    Science.gov (United States)

    Kozlov, D G; Cheperigin, S E; Chestkov, A V; Krylov, V N; Tsygankov, Iu D

    2010-03-01

    Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form. PMID:20391778

  7. High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

    Directory of Open Access Journals (Sweden)

    Hemant Kumar Verma

    Full Text Available Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.

  8. Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

    Science.gov (United States)

    Saito, Youhei; Nakagawa, Takanobu; Kakihana, Ayana; Nakamura, Yoshia; Nabika, Tomomi; Kasai, Michihiro; Takamori, Mai; Yamagishi, Nobuyuki; Kuga, Takahisa; Hatayama, Takumi; Nakayama, Yuji

    2016-09-01

    The mammalian stress protein Hsp105β, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105β-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105β- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105β, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc. PMID:26873636

  9. [The construction and the expression of V5 epitope fused human androgen receptor vector in the yeast cell].

    Science.gov (United States)

    Yang, Chen; Luo, Fangni; Dai, Weixing; Li, Shanshan; Huang, Renhua; Xie, Yangmei; Xue, Feiyue; Li, Xiangming

    2013-08-01

    When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD. The V5 epitope tag DNA with a 5'-BamH I and a 3'-EcoR I sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5. The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR. This fused vector was transformed into the yeast cell (W303-1A). Western blot was used to detect the V5 fused protein of AR, in the protocol of which the primary monoclonal antibody (IgG(2a)) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the specific protein in the given sample of the transformed yeast extract. The result showed that the fused protein of AR was expressed successfully in the yeast cell. PMID:24059072

  10. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast

    OpenAIRE

    Liu, Bo; Shi, DanYang; Chang, Shaohong; Gong, Xin; Yu, YunZhou; Sun, Zhiwei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. ...

  11. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe

  12. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    Science.gov (United States)

    Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

    2014-02-11

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

  13. A STUDY ON EXPRESSION OF ARmRNA IN HUMEN BPH TISSUE WITH IN SITU RT-PCR

    Institute of Scientific and Technical Information of China (English)

    邱曙东; 霍涌玮; 张秋养; 葛玲

    2002-01-01

    Objecive To check and compare the expression le vels of human androgen receptor (AR) mRNA in both normal adult prostate (NAP) an d benign prostate hyperplasia (BPH) tissues, and to try to find if BPH results f rom the abnormal transcription of ARmRNA in prostate. Methods Expression of the human ARmRNA in 14 paraffin-embedded prosta te tissues (4 cases of NAP and 10 cases of BPH) was studied with the direct in situ reverse transcription-polymerase chain reaction (RT-PC R). Quantitative analysis of the ARmRNA products was performed using the image a nalysis system. Results ①Specific ARmRNA was detected in bo th NAP and BPH speci mens and in both epithelia and interstitial cells. The positive products were re latively densely localized in the cytoplasm of perinuclear zone. ② The intensit y of ARmRNA signals in epithelial cells was significantly stronger than that in interstitial cells (P<0.001). However, there was no statistically significa nt difference in ARmRNA level between NAP and BPH; ③ The heterogeneity of ARmRN A signal and the androgen-independent cells were observed in prostatic epitheli a. Stronger positive signals of ARmRNA were shown in a few basal-cell layer (BC L) cells of BPH tissue, but were not found in that of NAP tissue. Conclusion Results of this study show that there is no signifi cant difference in the ARmRNA expression between NAP and BPH groups in both epit helium and interstitial cells. It may indicate that BPH does not result from the ARmRNA transcription in the prostate.

  14. A STUDY ON EXPRESSION OF ARmRNA IN HUMEN BPH TISSUE WITH IN SITU RT-PCR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objecive:To check and compare the expression levels of human androgen receptor(AR) mRNA in both normal adult prostate(NAP)and benign prostate hyperplasia(BPH) tissues,and to try to find if BPH results from the abnormal transcription of ARmRNA in prostate.Methods:Expression of the human ARmRNA in 14 paraffinembedded prostate tissues(4 cases of NAP and 10 cases of BPH)was studied with the direct in situ reverse transcription-polymerase chain reaction(RT-PCR).Quantitative analysis of the ARmRNA products was performed using the image analysis system.Results:(1)Specific ARmRNA was detected in both NAP and BPH specimens and in both epithelia and interstitial cells,The positive products were relatively densely localized in the cytoplasm of perinuclear zone.(2)The intensity of ARmRNA signals in epithelial cells was significantly stronger than that in interstitial cells(P<0.001).However,there was no statistically significant difference in ARmRNA level between NAP and BPH;(3)The heterogeneity of ARmRNA signal and the androgen-independent cells were observed in prostatic epithelia.Stronger positive signals of ARmRNA were shown in a few basal-cell layer(BCL) cells of BPH tissue,but were not found in that of NAP tissue,Conclusion:Results of this study show that there is no significant difference in the ARmRNA expression between NAP and BPH groups in both epithelium and interstitial cessl,It may indicate that BPH does not result from the ARmRNA transcription in the prostate.

  15. Selective inhibition of purified human phosphodiesterase 4A expressed in yeast cell GL62 by ciclamilast, piclamilast, and rolipram

    Institute of Scientific and Technical Information of China (English)

    Jun-chun CHEN; Ji-qiang CHEN; Qiang-min Xie; Yi-liang ZHU

    2004-01-01

    AIM: To improve the specific activity of human phosphodiesterase 4A (PDE4A) expressed in yeast cell GL62 and investigate the effects of selective phosphodiesterase 4 (PDE4) inhibitors (ciclamilast, piclamilast, and rolipram),selective phosphodiesterase 5 (PDES) inhibitor zaprinast, and cyclooxygenase (COX) inhibitors (aspirin, indomethacin)on human PDE4A activity expressed in yeast cell GL62. METHODS: Human PDE4A was expressed in yeast cell GL62 after CuSO4 induction and the specific activity of human PDE4A was improved by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. The activity of PDE4A was measured by high performance liquid chromatography (HPLC). RESULTS: Induced PDE4A activity expressed in crude yeast cell GL62 supernatant and pellet was (340±21) nmol.g-1.min-1 and (250±25) nmol.g-1.min-lrespectively. The specific activity of recombinant PDE4A in supernatant was improved 6.4 fold. Ciclamilast,piclamilast, and rolipram could inhibit PDE4A activity. The ICs0 values (95 % confidence limits) of ciclamilast,piclamilast, and rolipram were 1.27 (0.84-1.91), 66.4 (33.3-132.2), and 3.73 (2.51-5.53) μmol/L respectively.Zaprinast, aspirin, and indomethacin had no obvious inhibitory effect on PDE4A activity. CONCLUSION: The specific activity of PDE4A expressed in yeast cell GL62 can be improved by ammonium sulfate fractionation,DEAE Sephadex A-50 chromatography, and Sephadex G-100 chromatography. Ciclamilast, piclamilast, and rolipram can inhibit PDE4A activity while zaprinast, aspirin, and indomethacin have no obvious inhibitory effect on PDE4A activity. Human PDE4A expressed in GL62 might be useful in the research and screening of new selective PDE4 inhibitors.

  16. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts.

    Science.gov (United States)

    Bucarey, Sergio A; Noriega, Jorge; Reyes, Paulina; Tapia, Cecilia; Sáenz, Leonardo; Zuñiga, Alejandro; Tobar, Jaime A

    2009-09-25

    Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine. PMID:19664739

  17. Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation

    Directory of Open Access Journals (Sweden)

    Jian eZha

    2012-10-01

    Full Text Available Reducing xylitol formation is an important step in engineering xylose reductase/xylitol dehydrogenase pathway for ethanol production from xylose in Saccharomyces cerevisiae. In this study, to balance the expression of XYL1 and mutant XYL2 encoding xylose reductase and NADP+-preferring xylitol dehydrogenase for reduced xylitol production, we utilized a strategy combining chassis selection and direct fine-tuning of XYL1 and XYL2 expression. A XYL1 gene under control of various promoters of ADH1, truncated ADH1 and PGK1, and a mutated XYL2 with different copy numbers were constructed into different xylose-utilizing modules, which were then expressed in two yeast chassis W303a and L2612. The strategy enabled us to achieve an improved L2612-derived recombinant strain with XYL1 controlled by promoter PGK1 and with two copies of XYL2. The strain exhibited 21.3% lower xylitol yield and 40.0% higher ethanol yield. The results demonstrate the feasibility of the combinatorial strategy for construction of efficient xylose-fermenting Saccharomyces cerevisiae.

  18. Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

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    Passos Geraldo AS

    2006-08-01

    Full Text Available Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM, a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein, bgl (encoding for a 1,3-β-glucosidase in mycelium cells; and ags (an α-1,3-glucan synthase, cda (a chitin deacetylase and vrp (a verprolin in yeast cells; (ii ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken

  19. Zinc up-regulated the expression of the rice metallonthionein gene family and enhanced the zinc tolerance of yeast cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Northern blot and functional complementation assay were employed to analyze the effects of zinc on expression of ten rice metallothionein genes (OsMT-Is) in rice seedlings and the growth of yeast cells transformed with OsMT-Is. Northern blot revealed that in shoots of the rice seedlings treated with different Zn2+ concentrations, expression of most members of OsMT-I family was increased, except the type 4 OsMT-Is (OsMT-I-4a, 4b and 4c). In roots, Zn2+ significantly increased the transcription of OsMT-I-1b and OsMT-I-2c, but reduced the trascription of OsMT-I-1a and OsMT-I-3a. When these ten cDNAs were heterologously expressed in zinc sensitive yeast mutant, all transgenic yeasts showed increased tolerance to Zn2+, and zinc accumulation in these yeast cells also increased.These indicated that OsMT-I family members might respond to extra Zn2+, and they could enhance Zn2+ tolerance of cells by direct binding Zn2+.

  20. Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

    Science.gov (United States)

    Monsalve, R I; Lu, G; King, T P

    1999-08-01

    Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis). PMID:10425162

  1. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  2. Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast.

    Science.gov (United States)

    Jagadish, Krishnappa; Gould, Andrew; Borra, Radhika; Majumder, Subhabrata; Mushtaq, Zahid; Shekhtman, Alexander; Camarero, Julio A

    2015-07-13

    We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

  3. High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Ru-Shi Liu; Kun-Yu Yang; Jian Lin; Yi-Wei Lin; Zhi-Hong Zhang; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), theantigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN)protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P. pastoris) GS115 (HisMut+) by electroporation. His+Mut+recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His+Mut+ aftertransformation of P. pastoriswith linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN)protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 mL/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell ,A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P. pastoris was about 4-fold higher than that expressed in E.coliwhen the same rSCoVN protein

  4. A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter

    Directory of Open Access Journals (Sweden)

    Salcedo-Sora J

    2012-08-01

    Full Text Available Abstract Background Calcium (Ca2+ signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. Methods The Plasmodium falciparum Ca2+/H+ antiporter (PfCHA was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. Results Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies. Discussion The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent

  5. Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast

    Science.gov (United States)

    Sze, H.; Liang, F.; Hwang, I.; Curran, A. C.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes. Several Ca2+ pumps have been identified and characterized by functional expression of plant genes in a yeast mutant (K616). This expression system has opened the way to a genetic and biochemical characterization of the regulatory and catalytic features of diverse Ca2+ pumps. Plant Ca(2+)-ATPases fall into two major types: AtECA1 represents one of four or more members of the type IIA (ER-type) Ca(2+)-ATPases in Arabidopsis, and AtACA2 is one of seven or more members of the type IIB (PM-type) Ca(2+)-ATPases that are regulated by a novel amino terminal domain. Type IIB pumps are widely distributed on membranes, including the PM (plasma membrane), vacuole, and ER (endoplasmic reticulum). The regulatory domain serves multiple functions, including autoinhibition, calmodulin binding, and sites for modification by phosphorylation. This domain, however, is considerably diverse among several type IIB ATPases, suggesting that the pumps are differentially regulated. Understanding of Ca2+ transporters at the molecular level is providing insights into their roles in signaling networks and in regulating fundamental processes of cell biology.

  6. [The cloning and expression of the gene for beta-galactosidase from Candida pseudotropicalis yeasts in Saccharomyces cerevisiae cells].

    Science.gov (United States)

    Tretiak, K A; Zakal'skiĭ, A E; Gudz', S P

    1998-01-01

    The gene of beta-galactosidase of lactose-assimilating yeast Candida pseudotropicalis was cloned in pG2 and pBG2-3 hybrid shuttle vectors and expressed in Saccharomyces cerevisiae laboratory strains under the control of own promoter. The plasmids were able to replicate autonomously with relative stability in transformants of baker's yeasts. The availability of glucose or lactose in the medium influenced the recombinant plasmid stability and the expression of the cloned gene. A number of experiments have shown that the LAC+ phenotype in pG2-transformed Saccharomyces cerevisiae was due to the expression of the Candida pseudotropicalis lactose permease gene that is probably located in SaIG1/XhoI DNA fragment about 4.3 kb long. Southern hybridization experiments showed that LAC(+)-transformants of Saccharomyces cerevisiae contained both autonomously-replicative, and integrative pG2 plasmid.

  7. Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

    Science.gov (United States)

    Fossati, Sara Maria; Candiani, Simona; Nödl, Marie-Therese; Maragliano, Luca; Pennuto, Maria; Domingues, Pedro; Benfenati, Fabio; Pestarino, Mario; Zullo, Letizia

    2015-08-01

    Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

  8. D-amino acid-induced expression of D-amino acid oxidase in the yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Takahashi, Shouji; Okada, Hirotsune; Abe, Katsumasa; Kera, Yoshio

    2012-12-01

    We investigated D-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E. coli displayed oxidase activity to neutral and basic D-amino acids, but not to an L-amino acid or acidic D-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without D-amino acid, and was approximately doubled by adding D-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. L-Alanine also induced the activity, but only by about half of that induced by D-alanine. The induction by D-alanine reached a maximum level at 2 h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was D-alanine, followed by D-proline and then D-serine. Not effective were N-carbamoyl-D,L-alanine (a better inducer of DAO than D-alanine in the yeast Trigonopsis variabilis), and both basic and acidic D-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms. PMID:22986818

  9. Protective vaccination against infectious bursal disease virus with whole recombinant Kluyveromyces lactis yeast expressing the viral VP2 subunit.

    Directory of Open Access Journals (Sweden)

    Marina Arnold

    Full Text Available Here we report on vaccination approaches against infectious bursal disease (IBD of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis. Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV. Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.

  10. Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

    Science.gov (United States)

    Doyle, Patrick J; Saeed, Hanaa; Hermans, Anne; Gleddie, Steve C; Hussack, Greg; Arbabi-Ghahroudi, Mehdi; Seguin, Charles; Savard, Marc E; Mackenzie, C Roger; Hall, J Christopher

    2009-12-11

    15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops. PMID:19783651

  11. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds on both receptor types

    NARCIS (Netherlands)

    Bovee, T.F.H.; Helsdingen, J.R.; Rietjens, I.M.C.M.; Keijer, J.; Hoogenboom, L.A.P.

    2004-01-01

    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hER) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic

  12. Phenotypic expression of primary lesions of genetic material in Saccharomyces yeasts.

    Science.gov (United States)

    Inge-Vechtomov, S G; Repnevskaya, M V

    1989-01-01

    "Illegitimate" mating of yeasts (alpha x alpha), either spontaneous or induced by uv light or ethyl methanesulfanate, in a selective system for "cytoduction" revealed that about 95% of cytoductants expressed their original (alpha) mating type. Inducing the mating by treating the recipient of cytoplasm with uv light reached two orders of magnitude. An additional copy of MAT alpha in the alpha recipient almost completely eliminated the effect, which means that nonheritable mating type changes observed are formally recessive and are localized within MAT alpha complex. About 1% of cytoductants obtained were nonmating types and some of them were identified as mat alpha l mutants. Radl8 mutant as a recipient showed a considerably elevated spontaneous frequency of illegitimate hybridization and cytoduction. The cytoductants also preserved the original mating type. These facts suggest that nonheritable changes of mating type are due to repairable primary (premutational) lesions in MAT alpha genetic material. The significance of these results for understanding the mechanism of nonheritable variability is discussed.

  13. Expression of yellow jacket and wasp venom Ag5 allergens in bacteria and in yeast.

    Science.gov (United States)

    Monsalve, R I; Lu, G; King, T P

    1999-01-01

    Antigen 5 (Ag5), of unknown biological function, is one of the major venom allergens of vespids and fire ants. We have compared the expression of Ag5 in bacteria and in yeast. Recombinant Ag5 from bacteria formed an insoluble intracellular product, which was not properly folded, but that produced in Pichia pastoris was secreted to the extracellular medium. Immunochemical characterizations showed the secreted Ag5 to have the native structure of the natural protein. This is of interest since the B cell epitopes of Ag5 are mainly of the discontinuous type. These studies were made with Ag5s from yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis), and with hybrid Ag5 molecules that contained partial sequences of these two species. In vitro allergenicity studies with sera from yellow jacket-sensitive patients showed that some of these hybrid molecules had a greatly reduced allergenicity but retained the immunogenicity of the natural allergen. This could be of importance for immunotherapy of this type of allergy. PMID:11487873

  14. High-level expression of human tumour suppressor P53 in the methylotrophic yeast: Pichia pastoris.

    Science.gov (United States)

    Abdelmoula-Souissi, Salma; Rekik, Leila; Gargouri, Ali; Mokdad-Gargouri, Raja

    2007-08-01

    The human tumour suppressor P53 is a key protein involved in tumour suppression. P53 acts as a "guardian of genome" by regulating many target genes involved in cell cycle regulation, DNA repair and apoptosis. We report the P53 expression by the methylotrophic yeast Pichia pastoris using the methanol inducible AOX1 promoter. We have produced the rP53 in intracellular form as well as secreted using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence in two genetic contexts of Pichia, Mut(s) and Mut(+). The intracellular P53 was successfully produced by Mut(s) (KM71) as well as Mut(+) (X33) strains, however, the secreted form was mainly observed in the Mut(s) strain, despite a higher number of p53 copies integrated in the Mut(+) strain. Interestingly, in Mut(s) phenotype, the medium pH influences markedly the rP53 production since it was higher at pH 7 than 6. PMID:17482479

  15. Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

    Science.gov (United States)

    Hipp, Katharina; Schäfer, Benjamin; Kepp, Gabi; Jeske, Holger

    2016-01-01

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. PMID:27399762

  16. Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris.

    Science.gov (United States)

    Gu, Lina; Lajoie, Curtis; Kelly, Christine

    2003-01-01

    A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C. PMID:14524699

  17. Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Baumgartner, Philippe; Harper, Karen; Raemaekers, Romaan J M; Durieux, Alain; Gatehouse, Angharad M R; Davies, Howard V; Taylor, Mark A

    2003-08-01

    The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.

  18. Studies in the development of Japanese encephalitis vaccine: expression of virus envelope glycoprotein V3 (E) gene in yeast

    OpenAIRE

    Fujita, H; Sumiyoshi, H.; Mori, C.; Manabe, S.; Takagi, M.; Yoshida, I.; Morita, K.; Fuke, I.; FUKAI, K.; Igarashi, A.

    1987-01-01

    A safe, effective and economical vaccine is required for the prevention of Japanese encephalitis (JE), a disease with high mortality and grave sequelae, which is prevalent in Japan and other countries in east, south-east and southern Asia. As the initial step to produce a second-generation vaccine, recombinant DNA technology was utilized to express the JE virus envelope glycoprotein V3 (E) gene in yeast cells.

  19. Expression of human augmenter of liver regeneration in pichia pastoris yeast and its bioactivity in vitro

    Institute of Scientific and Technical Information of China (English)

    Qi Liu; Hui-Feng Yu; Hang Sun; Hua-Feng Ma

    2004-01-01

    AIM: To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro.METHODS: With PCR and gene recombination techniques,cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9. The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K. The recombinant plasmid pPIC9KhALR was transformed into GS115 with electroporation.hALR was expressed by GS1L5 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot. The effects of hALR on in vitro proliferation of QGY and HepG2 cells were evaluated by 3H-TdR methods.RESULTS: The correctness and integrity of recombinant plasmids ppIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods,respectively. hALR as a secretive protein was successfully expressed by GS115. Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid ppIC9K-hALR.The results of Western blot of hALR showed the specific band. The high qualitative hALR was obtained through ultrafiltration. hALR could stimulate in vitro proliferation of QGY and HepG2 cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG2.CONCLUSION: The hALR as a secretive protein can be successfully expressed by GS115. It may stimulate in vitro proliferation of QGY and HepG2 cells at a dose-dependent manner. But QGY and HepG2 cells have different reactivities to hALR.

  20. Detection of marine microalgal biotoxins using bioassays based on functional expression of tunicate xenobiotic receptors in yeast.

    Science.gov (United States)

    Richter, Ingrid; Fidler, Andrew E

    2015-03-01

    Marine microalgae can produce biotoxins that cause widespread poisoning in marine ecosystems and may also affect human health. While established microalgal biotoxins are detectable using chemical methods, a need remains for robust, inexpensive bioassays. Ligand-binding domains (LBDs) from a tunicate nuclear receptor, VDR/PXRα, which is orthologous to both the vertebrate pregnane X receptor (PXR) and the vitamin D receptor (VDR), can be activated by microalgal biotoxins when expressed in mammalian cell lines. Building on this observation, we developed a generic recombinant yeast bioassay platform that expresses chimeric proteins containing tunicate VDR/PXRα LBDs which mediate ligand-dependent transcription of a reporter gene (lacZ) encoding an easily assayed enzyme (β-galactosidase). Recombinant yeast strains expressing VDR/PXRα LBDs from two tunicate species, Ciona intestinalis and Botryllus schlosseri, were exposed to both synthetic and natural toxins. Structurally simple synthetic chemicals (n-butyl-p-aminobenzoate, carbamazepine, p-aminobenzoic acid, and bisphenol-A) generated EC50 values in the μM range, while more structurally complex marine biotoxins (okadaic acid, pectenotoxin-11, and portimine) activated the assays in the nM range. Given the large number of tunicate species, we propose that tunicate VDR/PXR LBDs may be used as 'sensor elements' in similar yeast-based high-throughput bioassays for detection of established microalgal biotoxins and uncharacterised marine bioactive compounds.

  1. Detection of marine microalgal biotoxins using bioassays based on functional expression of tunicate xenobiotic receptors in yeast.

    Science.gov (United States)

    Richter, Ingrid; Fidler, Andrew E

    2015-03-01

    Marine microalgae can produce biotoxins that cause widespread poisoning in marine ecosystems and may also affect human health. While established microalgal biotoxins are detectable using chemical methods, a need remains for robust, inexpensive bioassays. Ligand-binding domains (LBDs) from a tunicate nuclear receptor, VDR/PXRα, which is orthologous to both the vertebrate pregnane X receptor (PXR) and the vitamin D receptor (VDR), can be activated by microalgal biotoxins when expressed in mammalian cell lines. Building on this observation, we developed a generic recombinant yeast bioassay platform that expresses chimeric proteins containing tunicate VDR/PXRα LBDs which mediate ligand-dependent transcription of a reporter gene (lacZ) encoding an easily assayed enzyme (β-galactosidase). Recombinant yeast strains expressing VDR/PXRα LBDs from two tunicate species, Ciona intestinalis and Botryllus schlosseri, were exposed to both synthetic and natural toxins. Structurally simple synthetic chemicals (n-butyl-p-aminobenzoate, carbamazepine, p-aminobenzoic acid, and bisphenol-A) generated EC50 values in the μM range, while more structurally complex marine biotoxins (okadaic acid, pectenotoxin-11, and portimine) activated the assays in the nM range. Given the large number of tunicate species, we propose that tunicate VDR/PXR LBDs may be used as 'sensor elements' in similar yeast-based high-throughput bioassays for detection of established microalgal biotoxins and uncharacterised marine bioactive compounds. PMID:25549942

  2. Expression of Genes Related to Oxidative Stress in Yeast Treated with Ionizing Radiation and N-acetyl -L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Park, Ji Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-10-15

    Ionizing radiation (IR) induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage including DNA strand breaks (DSBs), base damage, protein damage and lipid-hydroperoxide. Detoxifying enzymes are immediately triggered for ROS scavenging. Yeast contains two forms of superoxide dismutase (SOD). SOD1 as a cytosolic copper-zinc superoxide dismutase is located in the cytoplasm and cytosol. SOD2 as a manganese containing enzyme is act in mitochondria matrix and mitochondrion. These enzymes scavenge superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast contains two catalases. Catalase A (CTA1) and Cytosolic catalase T (CTT1) is located in peroxisome and cytoplasm, respectively. Yeast has two glutathione (GSH) peroxidases, which are GPX1 and GPX2. GPX1 and GPX2 are component of cellular component and cytoplasm, respectively. The biochemical function of GSH peroxidase is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Otherwise, chemicals and materials help ROS detoxification against oxidative damage. N-acetyl-Lcysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. In this study, we examined the effect of NAC through gene expressions related to protective enzyme against oxidative stress in yeast

  3. Yeast surface display for directed evolution of protein expression, affinity, and stability.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    2000-01-01

    The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.

  4. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    Science.gov (United States)

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-01

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  5. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    OpenAIRE

    Woo Tae Park; Mariadhas Valan Arasu; Naif Abdullah Al-Dhabi; Sun Kyung Yeo; Jin Jeon; Jong Seok Park; Sook Young Lee; Sang Un Park

    2016-01-01

    The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L) and silver nitrate (30 mg/L) for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to t...

  6. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various GCN4-lacZ fusions, knockout yeast strains, and anti-eIF-2 -P/anti-eIF-2 antibodies, we observed that heterologous expression......, as a density of 1 of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation...

  7. Natural genetic variation impacts expression levels of coding, non-coding, and antisense transcripts in fission yeast

    DEFF Research Database (Denmark)

    Clément-Ziza, Mathieu; Marsellach, Francesc X.; Codlin, Sandra;

    2014-01-01

    Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated...... the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely...

  8. Heterologous expression of an insecticidal gene from the armed spider (Phoneutria nigriventer

    Directory of Open Access Journals (Sweden)

    J. E. F. Figueiredo

    2008-01-01

    Full Text Available Insect-pests are global problems that cause severe damage to crop plants, and their control is commonly based on chemical insecticides. However, negative effects of pesticides on the environment and human health emphasize the necessity to develop alternative methods for insect-pest control. In the present study, a gene coding for the insecticidal peptide TX4(6-1 of the Brazilian armed spider (Phoneutria nigriventer was cloned in fusion with maltose binding protein (MBP and expressed in Escherichia coli. The affinity purified protein MBP-GlyTX4 was cleaved with the Xa factor and used for a bioassay against Spodoptera frugiperda and rabbit immunization. Five micrograms GlyTX4 protein injected into the hemocoel of larvae and abdominal cavity of adults produced trembling and uncoordinated movements immediately after injection and all adult insects died after 12h. After two days, larvae became paralyzed and the epidermal color changed to dark brown. Furthermore, the development stage was prolonged for two weeks. Alternatively, slices of maize leaves were imbibed with 15 micrograms of the recombinant protein cleaved with the Xa factor and used as diet for larvae. In this experiment, all larvae died in about 30 minutes. Polyclonal antibodies anti-MBP-GlyTX4 were effective for recognizing MBP and GlyTX4 in whole cell extract from E. coli expressing the recombinant protein.

  9. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kun [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Sun, Guoxun [Department of Hematology, Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001 (China); Lv, Zhiyuan; Wang, Chen [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Jiang, Xueyuan, E-mail: xueyuanjiang@yahoo.com.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Li, Donghai, E-mail: lidonghai@gmail.com [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China); Zhang, Chenyu, E-mail: cyzhang@nju.edu.cn [Jiangsu Diabetes Research Center, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu (China)

    2010-10-01

    Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

  10. Direct evaluation of the effect of gene dosage on secretion of protein from yeast Pichia pastoris by expressing EGFP.

    Science.gov (United States)

    Liu, Hailong; Qin, Yufeng; Huang, Yuankai; Chen, Yaosheng; Cong, Peiqing; He, Zuyong

    2014-02-28

    Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression. PMID:24225373

  11. Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

    Science.gov (United States)

    Hamill, J D; Robins, R J; Parr, A J; Evans, D M; Furze, J M; Rhodes, M J

    1990-07-01

    Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation. PMID:2103440

  12. Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an -L-Rhamnosidase of enological interest

    OpenAIRE

    Manzanares, P.; Orejas, M; Vicente Gil, J.; Graaff, de, Laura Corina Geertruida; J. Visser; Ramon, D.

    2003-01-01

    The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a beta-glucosidase, show. increased content mainly of the aromatic compound linalool.

  13. Construction of a Genetically Modified Wine Yeast Strain Expressing the Aspergillus aculeatus rhaA Gene, Encoding an α-l-Rhamnosidase of Enological Interest

    OpenAIRE

    Manzanares, Paloma; Orejas, Margarita; Gil, José Vicente; de Graaff, Leo H; Visser, Jaap; Ramón, Daniel

    2003-01-01

    The Aspergillus aculeatus rhaA gene encoding an α-l-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a β-glucosidase, show increased content mainly of the aromatic compound linalool.

  14. Immune gene expression profile of Penaeus monodon in response to marine yeast glucan application and white spot syndrome virus challenge.

    Science.gov (United States)

    Wilson, Wilsy; Lowman, Douglas; Antony, Swapna P; Puthumana, Jayesh; Bright Singh, I S; Philip, Rosamma

    2015-04-01

    Immunostimulant potential of eight marine yeast glucans (YG) from Candida parapsilosis R20, Hortaea werneckii R23, Candida spencermartinsiae R28, Candida haemulonii R63, Candida oceani R89, Debaryomyces fabryi R100, Debaryomyces nepalensis R305 and Meyerozyma guilliermondii R340 were tested against WSSV challenge in Penaeus monodon post larvae (PL). Structural characterization of these marine yeast glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. PL were fed 0.2% glucan incorporated diet once in seven days for a period of 45 days and the animals were challenged with white spot syndrome virus (WSSV). The immunostimulatory activity of yeast glucans were assessed pre- and post-challenge WSSV by analysing the expression profile of six antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and 13 immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, caspase, catalase, glutathione peroxidase, glutathione-s-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO), Rab-7, superoxide dismutase and transglutaminase. Expression of seven WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, thymidine kinase and VP28 were also analysed to detect the presence and intensity of viral infection in the experimental animals post-challenge. The study revealed that yeast glucans (YG) do possess immunostimulatory activity against WSSV and also supported higher survival (40-70 %) post-challenge WSSV. Among the various glucans tested, YG23 showed maximum survival (70.27%), followed by YG20 (66.66%), YG28 (60.97%), YG89 (58.53%), YG100 (54.05%), YG63 (48.64%), YG305 (45.7%) and YG340 (43.24%). PMID:25555812

  15. Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.

    Science.gov (United States)

    Choi, Min-Yeon; Park, Sang-Hyun

    2016-06-01

    Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression. PMID:26837218

  16. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

    Directory of Open Access Journals (Sweden)

    Teixeira Miguel C

    2012-07-01

    Full Text Available Abstract Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC Superfamily and Major Facilitator Superfamily (MFS in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to

  17. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast.

    Science.gov (United States)

    Liu, Bo; Shi, DanYang; Chang, ShaoHong; Gong, Xin; Yu, YunZhou; Sun, ZhiWei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens. PMID:25567004

  18. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture.

    Science.gov (United States)

    Park, Woo Tae; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Yeo, Sun Kyung; Jeon, Jin; Park, Jong Seok; Lee, Sook Young; Park, Sang Un

    2016-01-01

    The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L) and silver nitrate (30 mg/L) for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  19. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

    Full Text Available The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L and silver nitrate (30 mg/L for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  20. Expression of the Salmonella spp. virulence factor SifA in yeast alters Rho1 activity on peroxisomes.

    Science.gov (United States)

    Vinh, Dani B N; Ko, Dennis C; Rachubinski, Richard A; Aitchison, John D; Miller, Samuel I

    2010-10-15

    The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing conditions, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25Δ and rho1 mutants, suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further definition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases.

  1. Thermotolerant yeasts selected by adaptive evolution express heat stress response at 30ºC

    DEFF Research Database (Denmark)

    Caspeta, Luis; Chen, Yun; Nielsen, Jens

    2016-01-01

    Exposure to long-term environmental changes across >100s of generations results in adapted phenotypes, but little is known about how metabolic and transcriptional responses are optimized in these processes. Here, we show that thermotolerant yeast strains selected by adaptive laboratory evolution ...

  2. Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast

    OpenAIRE

    Kim Kiat Lim; Terenze Yao Rui Ong; Yue Rong Tan; Eugene Guorong Yang; Bingbing Ren; Kwi Shan Seah; Zhe Yang; Tsu Soo Tan; Dymock, Brian W.; Ee Sin Chen

    2015-01-01

    Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibit...

  3. Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Hsien-Wei Meng

    Full Text Available SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (K(D  = ∼ 1 nM and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

  4. Analysis of expressed sequence tags from the anamorphic basidiomycetous yeast, Pseudozyma antarctica, which produces glycolipid biosurfactants, mannosylerythritol lipids.

    Science.gov (United States)

    Morita, Tomotake; Konishi, Masaaki; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

    2006-07-15

    Pseudozyma antarctica T-34 secretes a large amount of biosurfactants (BS), mannosylerythritol lipids (MEL), from different carbon sources such as hydrocarbons and vegetable oils. The detailed biosynthetic pathway of MEL remained unknown due to lack of genetic information on the anamorphic basidiomycetous yeasts, including the genus Pseudozyma. Here, in order to obtain genetic information on P. antarctica T-34, we constructed a cDNA library from yeast cells producing MEL from soybean oil and identified the genes expressed through the creation of an expressed sequence tags (EST) library. We generated 398 ESTs, assembled into 146 contiguous sequences. Based upon a BLAST search similarity cut-off of Eexpression study using real-time reverse transcriptase-PCR, the predicted genes, such as mannosyltranferase and acyltransferase, were demonstrated to be highly involved in MEL biosynthesis in soybean oil-grown cells. PMID:16845679

  5. Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast.

    Science.gov (United States)

    Lim, Kim Kiat; Ong, Terenze Yao Rui; Tan, Yue Rong; Yang, Eugene Guorong; Ren, Bingbing; Seah, Kwi Shan; Yang, Zhe; Tan, Tsu Soo; Dymock, Brian W; Chen, Ee Sin

    2015-01-01

    Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2(+) could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-A(cnp1-1) was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words]. PMID:26369364

  6. Rice OsRAD21-2 is Expressed in Actively Dividing Tissues and its Ectopic Expression in Yeast Results in Aberrant Cell Division and Growth

    Institute of Scientific and Technical Information of China (English)

    Chunyan Gong; Tang Li; Qi Li; Longfeng Yan; Tai Wang

    2011-01-01

    Rad21 and its meiotic counterpart Rec8,the key components of the cohesin complex,are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis,respectively.In contrast to yeast and vertebrates,which have only two RAD21/REC8 genes,the rice genome encodes four Rad21/Rec8 proteins.Here,we report on the cloning and characterization of OsRAD21-2 from rice (Oryza sativa L.).Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific Rad21 subfamily.Semi-quantitative reverse transcription-polymerase chain reaction revealed OsRAD21-2 preferentially expressed in premeiotic flowers.Further RNA in situ hybridization analysis and promoter::β-glucuronidase staining indicated that OsRAD21-2 was mainly expressed in actively dividing tissues including premeiotic stamen,stem intercalary meristem,leaf meristem,and root pericycle.Ectopic expression of OsRAD21-2 in fission yeast resulted in cell growth delay and morphological abnormality.Flow cytometric analysis revealed that the OsRAD21-2-expressed cells were arrested in G2 phase.Our results suggest that OsRad21-2 functions in regulation of cell division and growth.

  7. Regulatory concerns associated with use of value-added recombinant proteins and peptides screened in hgh-throughput for expression in fuel ethanol yeast strains

    Science.gov (United States)

    Recombinant proteins expressed in animals have been a public concern as a risk to the consumer since the animals are genetically modified to obtain desired improvements (GMO animals). Similarly, various commercially valuable proteins or peptides expressed in fuel ethanol yeast strains under develop...

  8. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression

    OpenAIRE

    Kühl, Inge; Dujeancourt, Laurent; Gaisne, Mauricette; Herbert, Christopher J.; Bonnefoy, Nathalie

    2011-01-01

    Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast...

  9. Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

    Science.gov (United States)

    Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

    2016-02-29

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis.

  10. Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer's yeast strain.

    Science.gov (United States)

    Wang, Jin-Jing; Wang, Zhao-Yue; He, Xiu-Ping; Zhang, Bo-Run

    2012-01-01

    Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product. PMID:22806798

  11. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan); Ohta, Miho [Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033 (Japan); Sakaki, Toshiyuki, E-mail: tsakaki@pu-toyama.ac.jp [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)

    2013-05-03

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.

  12. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform

    DEFF Research Database (Denmark)

    Mikkelsen, Michael Dalgaard; Buron, Line Due; Salomonsen, Bo;

    2012-01-01

    Epidemiological studies have shown that consumption of cruciferous vegetables, such as, broccoli and cabbages, is associated with a reduced risk of developing cancer. This phenomenon has been attributed to specific glucosinolates among the ∼30 glucosinolates that are typically present as natural...... products characteristic of cruciferous plants. Accordingly, there has been a strong interest to produce these compounds in microbial cell factories as it will allow production of selected beneficial glucosinolates. We have developed a versatile platform for stable expression of multi-gene pathways...... endogenous yeast activities with plant-derived enzymes. Production of indolylglucosinolate serves as a proof-of-concept for our expression platform, and provides a basis for large-scale microbial production of specific glucosinolates for the benefit of human health....

  13. Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

    DEFF Research Database (Denmark)

    Hansen, Klavs R.; Burns, G.; Mata, J.;

    2005-01-01

    sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr...... by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing....

  14. Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of α determinant region

    Institute of Scientific and Technical Information of China (English)

    Hui Hu; Xiao-Mou Peng; Yang-Su Huang; Lin Gu; Qi-Feng Xie; Zhi-Liang Gao

    2004-01-01

    AIM: Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.METHODS: HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD,respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA)and immune dot blotting to evaluate its antigenicity in vitro.The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.RESULTS: Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the α determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal antiHBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106±26.7 vs1 814.4±776.3, P<0.01,t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs6/10, 4.56±3.52 mIU/mL vs27.60±17.3 mIU/mL, P= 0.02, t= 2.7).CONCLUSIONS: HBsAg with deleted

  15. Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid

    International Nuclear Information System (INIS)

    The construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae is described. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transrormed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence or absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity. (Auth.)

  16. Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii

    Directory of Open Access Journals (Sweden)

    Plemenitaš Ana

    2007-08-01

    Full Text Available Abstract Background Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. Results A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. Conclusion Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at

  17. Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast

    Science.gov (United States)

    López-Torrejón, Gema; Jiménez-Vicente, Emilio; Buesa, José María; Hernandez, Jose A.; Verma, Hemant K.; Rubio, Luis M.

    2016-01-01

    The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals. PMID:27126134

  18. Design and characterization of a dual-mode promoter with activation and repression capability for tuning gene expression in yeast.

    Science.gov (United States)

    Mazumder, Mostafizur; McMillen, David R

    2014-08-01

    Modularity in controlling gene expression artificially is becoming an essential aspect of synthetic biology. Artificial transcriptional control of gene expression is one of the most well-developed methods for the design of novel synthetic regulatory networks. Such networks are intended to help understand natural cellular phenomena and to enable new biotechnological applications. Promoter sequence manipulation with cis-regulatory elements is a key approach to control gene expression transcriptionally. Here, we have designed a promoter that can be both activated and repressed, as a contribution to the library of synthetic biological 'parts'. Starting with the minimal cytochrome C (minCYC) promoter in yeast, we incorporated five steroid hormone responsive elements (SHREs) and one lac operator site, respectively, upstream and downstream of the TATA box. This allows activation through the testosterone-responsive androgen receptor, and repression through the LacI repressor. Exposure to varying concentrations of testosterone (to vary activation) and IPTG (to vary repression) demonstrated the ability to tune the promoter's output curve over a wide range. By integrating activating and repressing signals, the promoter permits a useful form of signal integration, and we are optimistic that it will serve as a component in future regulatory networks, including feedback controllers. PMID:25056312

  19. Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2)in methylotrophic yeast Pichia pastoris and its characterizations

    Institute of Scientific and Technical Information of China (English)

    YAN Xunyou; ZHAO Hongliang; ZHANG Weiguang; XUE Chong; LIU Zhimin

    2007-01-01

    To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.

  20. Existence and expression of photoreactivation repair genes in various yeast species

    International Nuclear Information System (INIS)

    Photoreactivation repair (Phr) activities in cell extracts of 13 different yeast species were measured by the Haemophilus influenzae transformation assay. Five species including Schizosaccharomyces pombe showed no or low enzymatic activity. In contrast to the other species, chromosomal DNAs of these 5 species did not show detectable hybridization using a DNA fragment of the photolyase PHRI gene of Saccharomyses cervisiae as a probe even at a low stringency condition. When the PHRI gene was attached to the 5'-flanking sequence of the iso-1-cytochrome c (CYC-1) gene of S. cerevisiae and introduced into S. pombe cells, the transformants acquired a high Phr activity, indicating that the PHR1 gene alone can provide a Phr-negative species with this repair activity and the light-absorbing cofactor(s) must be present in S. pombe. The results also demonstrated that the 5'-flanking sequence of the S. cervisiae and introduced into S. pombe cells, the transformants acquired a high Phr activity, indicating that the PHR gene alone can provide a Phr-negative species with this repair activity and the light-absorbing cofactor(s) must be present in S. pombe. The results also demonstrated that the 5'-flanking sequence of the S. cerevisiae CYC-1 gene works in S. pombe as a regulatory element. (author). 24 refs.; 4 figs.; 3 tabs

  1. Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast.

    Science.gov (United States)

    Bailey, M J; Siika-aho, M; Valkeajärvi, A; Penttilä, M E

    1993-02-01

    Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.

  2. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  3. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  4. Effects of N-acetyl-L-cysteine on gene expression of antioxidant enzymes in yeast cells after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu; Park, Ji Young; Ryu, Tae Ho; Roh, Chang Hyun [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2012-04-15

    Ionizing radiation induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage. When exposed to ionizing radiation, cells activates ROS scavenging detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. SOD scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast has two catalase and three GPx proteins. The biochemical function of GPx is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. N-acetylL-cysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity and alkaline phosphatase. In this study, the role of NAC as an antioxidant and a radioprotector was examined on cell survival, transcriptional level, and protein level. through observing viability of cells, analyzing the gene expression of antioxidant enzyme, measuring the SOD activity and intracellular GSH levels in yeast W303-1A strain The cell viability of haploid S. cerevisiae W303-1A strain was reduced significantly at the low dose (10∼30 Gy). The half-lethal dose of the strain was about 20 Gy. The CFU assay result confirmed that NAC could not rescue the cells from radiation-induced death. When irradiated with 100 Gy, an increase in the transcriptional expression was observed in the antioxicant genes. The expression of these genes decreased by treatment of NAC in irradiated cells. NAC decline SOD activity and intracellular GSH levels. The present study shows that NAC can directly scavenge

  5. Effect of Cottonseed Meal Fermented with Yeast on the Lipid-related Gene Expression in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    CX Nie

    2015-12-01

    Full Text Available ABSTRACT Fermented cottonseed meal (FCSM is widely used in poultry diets in China. This study was conducted to investigate the effect of FCSM on lipid-related gene expression in broilers. Initially, 180 broiler chickens (21-days-old, equal number of males and females were randomly divided into three groups, with six pens per group and 10 birds per pen. The chickens in the control group were fed a diet containing unfermented cottonseed meal, and those in the treatment groups were fed with diets including either CSM fermented by Candida tropicalis (Ct group or CSM fermented by Candida tropicalis plus Saccharomyces cerevisae (Ct-Sc group until 64 days old. The results revealed that, compared with the control group (p0.05. Likewise, the expressions of peroxisome proliferator-activated receptor gamma (PPAR-g and LPL in the abdominal fat were not altered by the FCSM-supplemented diets (p>0.05. The results in this study indicate that CSM fermented by Candida tropicalis and Saccharomyces cerevisiaeeffectively regulated the genes involved in fatty acid b-oxidation and triglyceride hydrolysis in male broiler chickens. Furthermore, the effects of the FCSM-supplemented diets were significantly different between bird sexes and between yeast strains used in the fermentation process.

  6. Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

    Directory of Open Access Journals (Sweden)

    Athmaram TN

    2011-11-01

    Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

  7. Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle.

    Science.gov (United States)

    Behera, Sthita Pragnya; Mishra, Niranjan; Nema, Ram Kumar; Pandey, Pooja Dubey; Kalaiyarasu, Semmannan; Rajukumar, Katherukamem; Prakash, Anil

    2015-01-01

    The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

  8. Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Wang, Tingting; Xu, Yongping; Liu, Wenjia; Sun, Yongxin; Jin, Liji

    2011-05-01

    Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture. PMID:21241808

  9. Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Cao Junxia(曹俊霞); Jin Liji; Duan Yanlong; An Lijia

    2003-01-01

    The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.

  10. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

    Science.gov (United States)

    Vassileva, A; Chugh, D A; Swaminathan, S; Khanna, N

    2001-06-01

    High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in

  11. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    Directory of Open Access Journals (Sweden)

    Claudia Husseneder

    Full Text Available The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival.

  12. Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites.

    Science.gov (United States)

    Husseneder, Claudia; Donaldson, Jennifer R; Foil, Lane D

    2016-01-01

    The Formosan subterranean termite, Coptotermes formosanus Shiraki, is a costly invasive urban pest in warm and humid regions around the world. Feeding workers of the Formosan subterranean termite genetically engineered yeast strains that express synthetic protozoacidal lytic peptides has been shown to kill the cellulose digesting termite gut protozoa, which results in death of the termite colony. In this study, we tested if Melittin, a natural lytic peptide from bee venom, could be delivered into the termite gut via genetically engineered yeast and if the expressed Melittin killed termites via lysis of symbiotic protozoa in the gut of termite workers and/or destruction of the gut tissue itself. Melittin expressing yeast did kill protozoa in the termite gut within 56 days of exposure. The expressed Melittin weakened the gut but did not add a synergistic effect to the protozoacidal action by gut necrosis. While Melittin could be applied for termite control via killing the cellulose-digesting protozoa in the termite gut, it is unlikely to be useful as a standalone product to control insects that do not rely on symbiotic protozoa for survival. PMID:26985663

  13. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  14. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

  15. Conformational change of glutathione-S-transferase by its co-expression with prion domain of yeast Ure2p

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The Ure2 protein from Saccharomyces cerevisisae has a changeable structure similar to that ofrnammalian prion protein. Its N-terminal is the prion domain (PrD) consisting of 65 amino acids which plays a critical role in yeast prion development. In this study, PrD gene was recombinated with glutathione-S-transferase(GST) gene, and a soluble GST-PrD(sGST-PrD) fusion protein was expressed in E. coli. sGST-PrD could spontaneously polymerize into amyloid fibrils in vitro, displaying typical β-sheet-type structure; it had increased resistance to proteinase K and exhibited amvloid-like optical properties. Moreover, the aggregated GST-PrD(aGST-PrD) could induce sGST-PrD to aggregate into fibrils. These results indicate that PrD could change the conformation of GST moiety in a recombinant protein with PrD to form a prion-like chimeric protein, which proves that PrD has the ability to mediate a prion-like conversion of other proteins fused with it.

  16. Definition of culture conditions for Arxula adeninivorans, a rational basis for studying heterologous gene expression in this dimorphic yeast.

    Science.gov (United States)

    Stöckmann, Christoph; Palmen, Thomas G; Schroer, Kirsten; Kunze, Gotthard; Gellissen, Gerd; Büchs, Jochen

    2014-06-01

    The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l(-1) was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l(-1). Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h(-1) and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.

  17. DNA Microspheres Coated with Bioavailable Polymer as an Efficient Gene Expression Agent in Yeasts

    Directory of Open Access Journals (Sweden)

    Irena Reytblat

    2016-01-01

    Full Text Available Gene delivery is one of the steps necessary for gene therapy and for genetic modification. However, delivering DNA into cells is challenging due to its negative charge that leads to repulsion by the negative cell membrane. In the current research, DNA spheres with a DNA encoding to a certain gene were coated with bioavailable polymers, polyethylene imine (PEI and polycaprolactone (PCL, in a short, one-step sonochemical reaction. The polymers were used in order to neutralize the negative charge of the DNA. Our study shows that the DNA nanospheres not only managed to penetrate the cell without causing it any damage, but also expressed the desired gene inside it.

  18. Heterologous expression and purification of barley (Hordeum vulgare L.) cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach;

    2011-01-01

    The mobilization of protein during germination of barley seeds is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction...... of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. The barley key cysteine protease, endoprotease...

  19. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui

    2004-01-01

    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  20. Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

    Directory of Open Access Journals (Sweden)

    Brooks Wynse S

    2011-09-01

    Full Text Available Abstract Background The trichothecene mycotoxin deoxynivalenol (DON may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON, and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201 were cloned and expressed in yeast (Saccharomyces cerevisiae during a series of small-scale ethanol fermentations using barley (Hordeum vulgare. DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

  1. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2013-02-12

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  2. Molecular Cloning and Yeast Expression of Cinnamate 4-Hydroxylase from Ornithogalum saundersiae Baker

    Directory of Open Access Journals (Sweden)

    Jian-Qiang Kong

    2014-01-01

    Full Text Available OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11, catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5′ non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD, HPLC-MS and nuclear magnetic resonance (NMR analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.

  3. [Expression of the Drosophila melanogaster limk1 gene 3'-UTRs mRNA in Yeast Saccharomyces cerevisiae].

    Science.gov (United States)

    Rumyantsev, A M; Zakharov, G A; Zhuravlev, A V; Padkina, M V; Savvateeva-Popova, E V; Sambuk, E V

    2014-06-01

    The stability of mRNA and its translation efficacy in higher eukaryotes are influenced by the interaction of 3'-untranscribed regions (3'-UTRs) with microRNAs and RNA-binding proteins. Since Saccharomyces cerevisiae lack microRNAs, it is possible to evaluate the contribution of only 3'-UTRs' and RNA-binding proteins' interaction in post-transcriptional regulation. For this, the post-transcriptional regulation of Drosophila limk1 gene encoding for the key enzyme of actin remodeling was studied in yeast. Analysis of limkl mRNA 3'-UTRs revealed the potential sites of yeast transcriptional termination. Computer remodeling demonstrated the possibility of secondary structure formation in limkl mRNA 3'-UTRs. For an evaluation of the functional activity of Drosophila 3'-UTRs in yeast, the reporter gene PHO5 encoding for yeast acid phosphatase (AP) fused to different variants of Drosophila limk1 mRNA 3'-UTRs (513, 1075, 1554 bp) was used. Assessments of AP activity and RT-PCR demonstrated that Drosophila limkl gene 3'-UTRs were functionally active and recognized in yeast. Therefore, yeast might be used as an appropriate model system for studies of 3'-UTR's role in post-transcriptional regulation.

  4. EV71 virus-like particles produced by co-expression of capsid proteins in yeast cells elicit humoral protective response against EV71 lethal challenge

    OpenAIRE

    Wang, Xiaowen; Xiao, Xiangqian; Zhao, Miao; Wei LIU; Pang, Lin; Sun, Xin; Cen, Shan; Burton B Yang; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Background Enterovirus 71 (EV71) is the most common causative pathogens of hand, foot and mouth disease (HFMD) associated with severe neurological complications. There is a great need to develop prophylactic vaccine against EV71 infection. Results EV71 virus-like particle (VLP) was produced in yeast expression system by the co-expression of four EV71 structural proteins VP1–VP4. Immunization with the recombinant VLPs elicited potent anti-EV71 antibody responses in adult mice and anti-VLP sera...

  5. Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

    Science.gov (United States)

    Cabrito, Tânia R; Teixeira, Miguel C; Duarte, Alexandra A; Duque, Paula; Sá-Correia, Isabel

    2009-10-01

    The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Deltatpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

  6. Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

    Directory of Open Access Journals (Sweden)

    Andrade-Garda José

    2008-04-01

    Full Text Available Abstract Background The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. Results We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Conclusion Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

  7. Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

    Science.gov (United States)

    Ståhlberg, Anders; Elbing, Karin; Andrade-Garda, José Manuel; Sjögreen, Björn; Forootan, Amin; Kubista, Mikael

    2008-01-01

    Background The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. Results We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Conclusion Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains. PMID:18412983

  8. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    Science.gov (United States)

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed. PMID:25239739

  9. Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.

    Science.gov (United States)

    Sonnewald, U; Brauer, M; von Schaewen, A; Stitt, M; Willmitzer, L

    1991-07-01

    In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis. PMID:1844880

  10. Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

    OpenAIRE

    Pérez-González, J. A.; González, R; Querol, A.; Sendra, J.; Ramón, D

    1993-01-01

    A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the...

  11. Differentially expressed genes under simulated deep-sea conditions in the psychrotolerant yeast Cryptococcus sp. NIOCCPY13

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, P.; Raghukumar, C.; Verma, A.K.; Meena, R.M.

    collected after exposure to the above mentioned three conditions using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Purification of polyA RNA was done using the polyA RNA purification kit (Clontech Laboratories, CA... The PCR-select SSH kit (Clontech Laboratories, CA, USA) was used to perform SSH on cDNA populations prepared from yeast cells according to the manufacturer’s instructions as follows: 1. Whole cell cDNA of the yeast subjected to 0.1 MPa/25º...

  12. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast.

    Science.gov (United States)

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D; Cho, Kyu Hong; Kim, Jeong-Ho

    2016-03-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor.

  13. Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

    Energy Technology Data Exchange (ETDEWEB)

    Fishel, L.A.; Villafranca, J.E.; Mauro, J.M.; Kraut, J.

    1987-01-27

    Using oligonucleotide-directed site-specific mutagenesis, they have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 /sup 0/C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

  14. Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

    International Nuclear Information System (INIS)

    Using oligonucleotide-directed site-specific mutagenesis, they have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 0C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability

  15. Differential transcription-activating capability of NS1 proteins from different influenza virus subtypes expressed in yeast

    Institute of Scientific and Technical Information of China (English)

    LI WeiZhong; WANG GeFei; ZENG Jun; ZHANG DanGui; ZHANG Heng; CHEN XiaoXuan; CHEN Ying; Li KangSheng

    2009-01-01

    Influenza A virus NS1 protein is an important regulatory factor with multiple functions and contributes greatly to viral pathogenesis. In the present study, transcription-activating potential of NS1 from dif-ferent influenza A virus subtypes was examined in yeast two-hybrid system. The bait vectors contain-ing different NS1 genes, along with an empty prey vector, were transformed into yeast AH109(for growth assay on QDO plate and a-galactosidase assay), and Y187(for β-galactosidase assay). AH109transformants with NS1 gene from H1N1, H5N1, and HgN2 viruses grew vigorously on the QDO plate and secreted high level of a-galactosidase. Also, Y187 bearing the above NS1 genes exhibited en-hanced β-galactosidase activity. Nevertheless, H3N2-NSl-transformed AH109 and Y187 yeasts did not grow on QDO plate and secrete β-galactosidase, respectively. These findings denote the remarkable variation in NS1 proteins from different influenza A virus subtypes on the transcription-stimulating capability in yeast.

  16. Differential transcription-activating capability of NS1 proteins from different influenza virus subtypes expressed in yeast

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Influenza A virus NS1 protein is an important regulatory factor with multiple functions and contributes greatly to viral pathogenesis.In the present study,transcription-activating potential of NS1 from different influenza A virus subtypes was examined in yeast two-hybrid system.The bait vectors contain-ing different NS1 genes,along with an empty prey vector,were transformed into yeast AH109(for growth assay on QDO plate and α-galactosidase assay),and Y187(for β-galactosidase assay).AH109 transformants with NS1 gene from H1N1,H5N1,and H9N2 viruses grew vigorously on the QDO plate and secreted high level of α-galactosidase.Also,Y187 bearing the above NS1 genes exhibited en-hanced β-galactosidase activity.Nevertheless,H3N2-NS1-transformed AH109 and Y187 yeasts did not grow on QDO plate and secrete β-galactosidase,respectively.These findings denote the remarkable variation in NS1 proteins from different influenza A virus subtypes on the transcription-stimulating capability in yeast.

  17. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina;

    2004-01-01

    Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transfer...

  18. Partially Functional Outer-Arm Dynein in a Novel Chlamydomonas Mutant Expressing a Truncated γ Heavy Chain▿

    Science.gov (United States)

    Liu, Zhongmei; Takazaki, Hiroko; Nakazawa, Yuki; Sakato, Miho; Yagi, Toshiki; Yasunaga, Takuo; King, Stephen M.; Kamiya, Ritsu

    2008-01-01

    The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain α and β heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the γ heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the α heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein. PMID:18487347

  19. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast.

    OpenAIRE

    McLeod, M; Stein, M.; Beach, D

    1987-01-01

    In fission yeast the ability to undergo meiosis and sporulation is conferred by the matP+ and matM+ genes of the mating-type locus. Inactivation of ran1+, a negative regulator of meiosis, is thought to be an essential step in meiotic initiation. We have isolated a further meiotic control gene mei3+, and have shown the following: a null allele of mei3 totally inhibits meiosis; the mei3+ RNA transcript and its translational product are expressed only in matP+/matM+ diploids entering meiosis; fo...

  20. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  1. Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system on the human tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expression that induces Rb gene inactivation and animal cells immortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eucaryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of column chromatography on HiTrap Q and HiTrap SP. The purified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.

  2. Recombinant expression of a GH12 β-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.

    Science.gov (United States)

    Picart, Pere; Orejas, Margarita; Pastor, F I Javier

    2016-08-01

    The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete. PMID:27339304

  3. Forces in yeast flocculation

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

    2015-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  4. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Kunkel Thomas A

    2005-06-01

    Full Text Available Abstract Background Antibody genes are diversified by somatic hypermutation (SHM, gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID. According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base excision repair. The DNA sequence context of mutation hotspots at G-C pairs during SHM is DGYW/WRCH (G-C is a hotspot position, R = A/G, Y = T/C, W = A/T, D = A/G/T. Results To investigate the mechanisms of AID-induced mutagenesis in a model system, we studied the genetic consequences of AID expression in yeast. We constructed a yeast vector with an artificially synthesized human AID gene insert using codons common to highly expressed yeast genes. We found that expression of the artificial hAIDSc gene was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain. A majority of mutations were at G-C pairs. In the ung1 strain, C-G to T-A transitions were found almost exclusively, while a mixture of transitions with 12% transversions was characteristic in the wild-type strain. In the ung1 strain mutations that could have originated from deamination of the transcribed stand were found more frequently. In the wild-type strain, the strand bias was reversed. DGYW/WRCH motifs were preferential sites of mutations. Conclusion The results are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. The sequence contexts of mutations in yeast induced by AID and those of somatic mutations at G-C pairs in immunoglobulin genes are significantly similar. This indicates that the intrinsic substrate specificity of AID itself is a primary determinant of mutational hotspots at G

  5. Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

    Science.gov (United States)

    Sahu, Umakant; Rangarajan, Pundi N

    2016-09-23

    Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris.

  6. Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

    Science.gov (United States)

    Sahu, Umakant; Rangarajan, Pundi N

    2016-09-23

    Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases, respectively; and (iv) GLN1 encoding glutamine synthetase. Synthesis of all these enzymes is regulated by Mxr1p at the level of transcription except GDH2, whose synthesis is regulated at the level of translation. Mxr1p activates the transcription of AAT1, AAT2, and GLN1 in cells cultured in YP as well as in YNB + Glu media, whereas transcription of MDH1 and MDH2 is activated in cells cultured in YNB + Glu but not in YP. A truncated Mxr1p composed of 400 N-terminal amino acids activates transcription of target genes in cells cultured in YP but not in YNB + Glu. Mxr1p binds to Mxr1p response elements present in the promoters of AAT2, MDH2, and GLN1 We conclude that Mxr1p is essential for utilization of amino acids as the sole source of carbon and nitrogen, and it is a global regulator of multiple metabolic pathways in P. pastoris. PMID:27519409

  7. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  8. 31P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

    International Nuclear Information System (INIS)

    Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creating kinase activities similar to those found in mammalian heart muscle. 31P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts

  9. Production of l-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases

    OpenAIRE

    Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Rajgarhia, Vineet; Suominen, Pirkko; Penttilä, Merja

    2013-01-01

    Background Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding l-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expre...

  10. Strategies for improving production and purification of a recombinant protein: rP30 of Toxoplasma gondii expressed in the yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Rolland, D; Raymond, F; Gauthier, M; Fournier, C; Charrier, J P; Jolivet, M; Dantigny, P

    2008-01-15

    Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

  11. The Physiological and Biochemical Mechanisms Providing the Increased Constitutive Cold Resistance in the Potato Plants, Expressing the Yeast SUC2 Gene Encoding Apoplastic Invertase

    Directory of Open Access Journals (Sweden)

    A.N. Deryabin

    2016-05-01

    Full Text Available The expression of heterologous genes in plants is an effective method to improve our understanding of plant resistance mechanisms. The purpose of this work was to investigate the involvement of cell-wall invertase and apoplastic sugars into constitutive cold resistance of potato (Solanum tuberosum L., cv. Dйsirйe plants, which expressed the yeast SUC2 gene encoding apoplastic invertase. WT-plants of a potato served as the control. The increase in the essential cell-wall invertase activity in the leaves of transformed plants indicates significant changes in the cellular carbohydrate metabolism and regulatory function of this enzyme. The activity of yeast invertase changed the composition of intracellular sugars in the leaves of the transformed potato plant. The total content of sugars (sucrose, glucose, fructose in the leaves and apoplast was higher in the transformants, in comparison by WT-plants. Our data indicate higher constitutive resistance of transformants to severe hypothermia conditions compared to WT-plants. This fact allows us to consider cell-wall invertase as a enzyme of carbohydrate metabolism playing an important regulatory role in the metabolic signaling upon forming increased plant resistance to low temperature. Thus, the potato line with the integrated SUC2 gene is a convenient tool to study the role of the apoplastic invertase and the products of its activity during growth, development and formation constitutive resistance to hypothermia.

  12. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    Science.gov (United States)

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions. PMID:16366681

  13. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters.

    Science.gov (United States)

    Verstrepen, Kevin J; Van Laere, Stijn D M; Vanderhaegen, Bart M P; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S; Winderickx, Joris; Thevelein, Johan M; Delvaux, Freddy R

    2003-09-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes. PMID:12957907

  14. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin.

    Science.gov (United States)

    Liu, Xiao-Yan; Chi, Zhe; Liu, Guang-Lei; Madzak, Catherine; Chi, Zhen-Ming

    2013-02-01

    In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

  15. The Freedom of Expression of Members of the Armed Forces Under the European Convention on Human Rights In Jokšas V. Lithuania

    Directory of Open Access Journals (Sweden)

    Kirchner Stefan

    2014-06-01

    Full Text Available Freedom of expression is one of the most fundamental rights in a democratic society. In fact, the freedom to express one’s opinion and to impart, as well as to receive, information, is essential for the participation in the democratic process. The ability to make decisions as a citizen requires access to information; the participation in the life of the society requires the ability to express one’s opinions. It is imperative that in a democratic society, as it is envisaged by the European Convention on Human Rights (ECHR, everybody is able to express their views, regardless as to whether these views correspond to the views of those who are in power. This ability is one of the key differences between democracy anddictatorship. In particular in the nation-states of Eastern Europe, which have only known freedom for a bit less than a quarter of a century, the growth of democratic structures is inextricably linked to the ability to exercise this right. But while human rights in principle pit the citizen against the State, the citizen who serves the State in a professional function might also wish to express opinions that go against the view of those who are entrusted with leading the State. This is particularly the case when it comes to members of the armed forces. The jurisprudence of the Convention organs with regard to the right of public officials and other State agents to express their opinion freely is not as coherent as it is with regard to other questions concerning the ECHR. In a case decided in late 2013, the European Court of Human Rights dealt with this question with regard to Lithuania. In this article, the authors look at the question of how far the State can restrict the freedom of expression of members of the armed forces under the European Convention on Human Rights.

  16. Expression, purification, crystallization and preliminary phasing of the heteromerization domain of the tRNA-export and aminoacylation cofactor Arc1p from yeast

    International Nuclear Information System (INIS)

    The heteromerization domain of an aminoacyl-tRNA synthetase cofactor from yeast was crystallized, complete selenomethionine MAD data were collected to 2.8 Å resolution and preliminary phasing reveals the presence of 20 monomers in the asymmetric unit. Eukaryotic aminoacyl-tRNA synthetases (aaRSs) must be integrated into an efficient tRNA-export and shuttling machinery. This is reflected by the presence of additional protein–protein interaction domains and a correspondingly higher degree of complex formation in eukaryotic aaRSs. However, the structural basis of interaction between eukaryotic aaRSs and associated protein cofactors has remained elusive. The N-terminal heteromerization domain of the tRNA aminoacylation and export cofactor Arc1p has been cloned from yeast, expressed and purified. Crystals have been obtained belonging to space group C2, with unit-cell parameters a = 222.32, b = 89.46, c = 126.79 Å, β = 99.39°. Calculated Matthews coefficients are compatible with the presence of 10–25 monomers in the asymmetric unit. A complete multiple-wavelength anomalous dispersion data set has been collected from a selenomethionine-substituted crystal at 2.8 Å resolution. Preliminary phasing reveals the presence of 20 monomers organized in five tetramers per asymmetric unit

  17. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

    Science.gov (United States)

    Wu, Dianhui; Li, Xiaomin; Lu, Jian; Chen, Jian; Zhang, Liang; Xie, Guangfa

    2016-01-01

    Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine.

  18. Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

    Science.gov (United States)

    Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

    2010-01-01

    In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

  19. Production of Candida antaractica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell & Expression in Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production

    Science.gov (United States)

    A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was produced using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. The lycotoxin-1 (Lyt-1) C3 variant gene ORF was added in-frame with the CALB ORF to pote...

  20. Robotic arm

    Science.gov (United States)

    Kwech, Horst

    1989-04-18

    A robotic arm positionable within a nuclear vessel by access through a small diameter opening and having a mounting tube supported within the vessel and mounting a plurality of arm sections for movement lengthwise of the mounting tube as well as for movement out of a window provided in the wall of the mounting tube. An end effector, such as a grinding head or welding element, at an operating end of the robotic arm, can be located and operated within the nuclear vessel through movement derived from six different axes of motion provided by mounting and drive connections between arm sections of the robotic arm. The movements are achieved by operation of remotely-controllable servo motors, all of which are mounted at a control end of the robotic arm to be outside the nuclear vessel.

  1. Yeast Interacting Proteins Database: YFR015C, YFR015C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...ression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into stationary ...tion, nitrogen starvation, environmental stress, and entry into stationary phase Rows with this bait as bait..., the more highly expressed yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental

  2. The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus.

    Science.gov (United States)

    Hua, Sui Sheng T; Beck, John J; Sarreal, Siov Bouy L; Gee, Wai

    2014-05-01

    Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus. PMID:24504634

  3. Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

    Directory of Open Access Journals (Sweden)

    Mitsuyoshi Ueda

    2013-09-01

    Full Text Available Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

  4. 酵母胱硫醚β-合成酶的表达及酶活鉴定%Expression and Characterization of Yeast Cystathionine β-Synthase

    Institute of Scientific and Technical Information of China (English)

    王利群; 奚学志; 曹利民; 顾雅萍; 孙培培

    2013-01-01

    将重组质粒pET45b-yCBS转入E.coli BL21中,构建高效表达酵母胱硫醚β-合成酶(yeast cystathionine β-synthase,1)的重组菌.研究诱导时菌体浓度、诱导剂IPTG浓度、诱导时间和温度,以及在培养基中添加不同浓度的山梨醇、葡萄糖、甘油对1表达量的影响.使用Ni2+亲和色谱柱纯化重组蛋白,再经His-Trap脱盐柱脱盐,以茚三酮法检测蛋白活性.结果表明,培养基中添加0.05%的山梨醇,重组菌在37℃培养3h后,添加终浓度为0.1 mmo/L的IPTG于20℃诱导培养15h,可溶性1表达量达到110 mg/L;纯化后比活为1 320 u/mg.%The recombinant vector pET45b-yCBS was constructed and transformed into E.coli BL21 to over-express yeast cystathionine β-synthase (1).The effects of various factors on the protein expression were studied,including strain density,the concentration of the inducer IPTG and inducing temperature and time,as well as the cosubstrates such as sorbitol,glucose and glycerin.The expressed protein was purified by Ni2+ chelating affinity chromatography and desalted by His-Trap desalting column,and its activity was determined with the ninhydrin reaction.The results indicated that the expression level of 1 is the highest when the recombinant strain was induced by 0.1 mmol/L IPTG for 15 h at 20 ℃ after cultured for 3 h at 37 ℃ in the LB medium supplemented with 0.05 % sorbitol.The productivity of the soluble 1 reached 110 mg/L.The specific activity of purified 1 is 1 320 u/mg.

  5. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance, gut morphology, and gene expression of pattern recognition receptors and cytokines in broiler chickens.

    Science.gov (United States)

    Alizadeh, M; Rodriguez-Lecompte, J C; Rogiewicz, A; Patterson, R; Slominski, B A

    2016-03-01

    An experiment was carried out to investigate the effect of yeast-derived products and distillers' dried grains with solubles (DDGS) on growth performance, small intestinal morphology, and innate immune response in broiler chickens from 1 to 21 d of age. Nine replicates of 5 birds each were assigned to dietary treatments consisting of a control diet without antibiotic (C), and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus, 0.025% of nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On d 21, 5 birds per treatment were euthanized and approximately 5-cm long duodenum, jejunum, and ileum segments were collected for intestinal morphology measurements. Cecal tonsils and spleen were collected to measure the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, macrophage mannose receptor (MMR), and cytokines IFN-γ, IL-12, IL-10, and IL-4. No significant difference was observed for growth performance parameters. However, diets containing 0.05% of nucleotides and YCW significantly increased (P < 0.05) villus height in the jejunum. Furthermore, the number of the goblet cells per unit area in the ileum was increased (P < 0.05) in diets supplemented with yeast-derived products. The expression of TLR2b in the spleen was down-regulated for diets supplemented with nucleotides and antibiotic. In addition, lower expression of TLR21 and MMR was observed in the spleen of birds receiving yeast-derived products and antibiotic. However, expression of TLR4 in the spleen was up-regulated in diets supplemented with YCW and nucleotides. The expression of IFN-γ and IL-12 was down-regulated in the spleen of birds fed diets supplemented with yeast-derived products. In addition, inclusion of YCW, Maxi-Gen Plus, or 0.05% of nucleotides down-regulated the expression of IL-10 and IL-4 in the cecal tonsils. In conclusion, down-regulation of receptors and cytokines in spleen and cecal tonsils of

  6. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  7. Discovering Aptamers by Cell-SELEX against Human Soluble Growth Factors Ectopically Expressed on Yeast Cell Surface

    OpenAIRE

    Hsien-Wei Meng; Pagano, John M.; White, Brian S.; Yoshiko Toyoda; Min, Irene M.; Craighead, Harold G; David Shalloway; Lis, John T.; Kai Xiao; Moonsoo M Jin

    2014-01-01

    SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on...

  8. Responses of transgenic Arabidopsis plants and recombinant yeast cells expressing a novel durum wheat manganese superoxide dismutase TdMnSOD to various abiotic stresses.

    Science.gov (United States)

    Kaouthar, Feki; Ameny, Farhat-Khemakhem; Yosra, Kamoun; Walid, Saibi; Ali, Gargouri; Faiçal, Brini

    2016-07-01

    In plant cells, the manganese superoxide dismutase (Mn-SOD) plays an elusive role in the response to oxidative stress. In this study, we describe the isolation and functional characterization of a novel Mn-SOD from durum wheat (Triticum turgidum L. subsp. Durum), named TdMnSOD. Molecular phylogeny analysis showed that the durum TdMnSOD exhibited high amino acids sequence identity with other Mn-SOD plants. The three-dimensional structure showed that TdMnSOD forms a homotetramer and each subunit is composed of a predominantly α-helical N-terminal domain and a mixed α/β C-terminal domain. TdMnSOD gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdMnSOD enhances tolerance of the transformed yeast cells to salt, osmotic, cold and H2O2-induced oxidative stresses. Moreover, the analysis of TdMnSOD transgenic Arabidopsis plants subjected to different environmental stresses revealed low H2O2 and high proline levels as compared to the wild-type plants. Compared with the non-transformed plants, an increase in the total SOD and two other antioxidant enzyme activities including catalase (CAT) and peroxidases (POD) was observed in the three transgenic lines subjected to abiotic stress. Taken together, these data provide evidence for the involvement of durum wheat TdMnSOD in tolerance to multiple abiotic stresses in crop plants. PMID:27152457

  9. Interaction Between Yeasts and Zinc

    Science.gov (United States)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  10. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    Science.gov (United States)

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

  11. Arm-Gal4 inheritance influences development and lifespan in Drosophila melanogaster.

    Science.gov (United States)

    Slade, F A; Staveley, B E

    2015-01-01

    The UAS-Gal4 ectopic expression system is a widely used and highly valued tool that allows specific gene expression in Drosophila melanogaster. Yeast transcription factor Gal4 can be directed using D. melanogaster transcriptional control elements, and is often assumed to have little effect on the organism. By evaluation of the consequences of maternal and paternal inheritance of a Gal4 transgene under the transcriptional regulation of armadillo control elements (arm-Gal4), we demonstrated that Gal4 expression could be detrimental to development and longevity. Male progeny expressing arm-Gal4 in the presence of UAS-lacZ transgene had reduced numbers and size of ommatidia, compared to flies expressing UAS-lacZ transgene under the control of other Gal4 transgenes. Aged at 25°C, the median life span of male flies with maternally inherited elav-Gal4 was 70 days, without a responding transgene or with UAS-lacZ. The median life span of maternally inherited arm-Gal4 male flies without a responding transgene was 48 days, and 40 days with the UAS-lacZ transgene. A partial rescue of this phenotype was observed with the expression of UAS-lacZ under paternal arm-Gal4 control, having an average median lifespan of 60 days. This data suggests that arm-Gal4 has detrimental effects on Drosophila development and lifespan that are directly dependent upon parental inheritance, and that the benign responder and reporter gene UAS-lacZ may influence D. melanogaster development. These findings should be taken into consideration during the design and execution of UAS-Gal4 expression experiments. PMID:26505429

  12. Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

    Science.gov (United States)

    Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

    2003-07-11

    Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

  13. Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

    Directory of Open Access Journals (Sweden)

    Alene Kast

    2015-05-01

    Full Text Available Cytoplasmic virus like elements (VLEs from Kluyveromyces lactis (Kl, Pichia acaciae (Pa and Debaryomyces robertsiae (Dr are extremely A/T-rich (>75% and encode toxic anticodon nucleases (ACNases along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5 results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

  14. Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

    Science.gov (United States)

    Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

    2015-05-01

    Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle. PMID:25973601

  15. Influence of N-glycans on Expression of Cell Wall Remodeling Related Genes in Paracoccidioides brasiliensis Yeast Cells

    Science.gov (United States)

    Almeida, Fausto; Antoniêto, Amanda Cristina Campos; Pessoni, André Moreira; Monteiro, Valdirene Neves; Alegre-Maller, Ana Claudia Paiva; Pigosso, Laurine Lacerda; Pereira, Maristela; Soares, Célia Maria de Almeida; Roque-Barreira, Maria Cristina

    2016-01-01

    Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as β-1,3-glucanosyltransferase (PbGEL3), 1,3-β-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-β-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and β-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM. PMID:27226767

  16. Evaluation of antibody response in mice against avian influenza A (H5N1) strain neuraminidase expressed in yeast Pichia pastoris

    Indian Academy of Sciences (India)

    Murugan Subathra; Ponsekaran Santhakumar; Mangamoori Lakshmi Narasu; Syed Sultan Beevi; Sunil K Lal

    2014-06-01

    Avian influenza has raised many apprehension in the recent years because of its potential transmitability to humans. With the increasing emergence of drug-resistant avian influenza strains, development of potential vaccines are imperative to manage this disease. Two structural antigens, haemagglutinin and neuraminidase, have been the target candidates for the development of subunit vaccine against influenza. In an effort to develop a faster and economically beneficial vaccine, the neuraminidase gene of a highly pathogenic avian influenza isolate was cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant neuraminidase (rNA) antigen was purified, and its bioactivity was analysed. The rNA was found to be functional, as determined by the neuraminidase assay. Four groups of mice were immunized with different concentrations of purified rNA antigen, which were adjuvanted with aluminium hydroxide. The immune response against rNA was analysed by enzyme-linked immunosorbent assay (ELISA) and neuraminidase inhibition assay. The mice groups immunized with 25 g and 10 g of antigen had a significant immune response against rNA. This method can be utilized for faster and cost-effective development of vaccines for a circulating and newer strain of avian influenza, and would aid in combating the disease in a pandemic situation, in which production time matters greatly.

  17. Regulation of unbalanced redox homeostasis induced by the expression of wild-type HIV-1 viral protein R (NL4-3Vpr) in fission yeast.

    Science.gov (United States)

    Gazdag, Zoltán; Stromájer-Rácz, Timea; Belagyi, Joseph; Zhao, Richard Y; Elder, Robert T; Virág, Eszter; Pesti, Miklós

    2015-09-01

    The wild-type viral protein R (Vpr) of human immunodeficiency virus type 1 exerts multiple effects on cellular activities during infection, including the induction of cell cycle G2 arrest and the death of human cells and cells of the fission yeast Schizosaccharomyces pombe. In this study, wild-type Vpr (NL4-3Vpr) integrated as a single copy gene in S. pombe chromosome was used to investigate the molecular impact of Vpr on cellular oxidative stress. NL4-3Vpr triggered an atypical response in early (14-h), and a wellregulated oxidative stress response in late (35-h) log-phase cultures. Specifically, NL4-3Vpr expression induced oxidative stress in the 14-h cultures leading, to decreased levels of superoxide anion (O(2)(·-)), hydroxyl radical (·OH) and glutathione (GSH), and significantly decreased activities of catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase. In the 35-h cultures, elevated levels of O(2)(·-) and peroxides were accompanied by increased activities of most antioxidant enzymes, suggesting that the Vpr-induced unbalanced redox state of the cells might contribute to the adverse effects in HIV-infected patients. PMID:26344028

  18. Yeast Genetics and Biotechnological Applications

    Science.gov (United States)

    Mishra, Saroj; Baranwal, Richa

    Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast Saccharomyces cerevisiae has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 μm plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.

  19. Yeast Interacting Proteins Database: YFR015C, YLR258W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into statio

  20. Integrated copy number and expression analysis identifies profiles of whole-arm chromosomal alterations and subgroups with favorable outcome in ovarian clear cell carcinomas.

    Directory of Open Access Journals (Sweden)

    Yuriko Uehara

    Full Text Available Ovarian clear cell carcinoma (CCC is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs and endometrioid carcinomas (ECs are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs. We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs, significantly lower than for SCs (93%, P < 0.01 and ECs (50%, P = 0.017. The ratio of whole-arm to all CNAs was higher in CCCs (46.9% than SCs (21.7%; P < 0.0001. SCs with loss of heterozygosity (LOH of BRCA1 (85% also had LOH of NF1 and TP53, and LOH of BRCA2 (62% coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10 showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041. Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64% than in CCC-2 (0/10, 0%; P < 0.01. Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.

  1. Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo.

    Science.gov (United States)

    Patterson, Robert; Eley, Thomas; Browne, Christopher; Martineau, Henny M; Werling, Dirk

    2015-11-17

    Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV-associated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a "proof of concept" vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of viraemia in sera and faeces of unvaccinated pigs was higher than that of vaccinated pigs. Additionally more sIgA was found in faeces of vaccinated pigs than unvaccinated. Vaccination was associated with lower serum concentrations of TNFα and IL-1β but higher concentrations of IFNα and IFNγ in comparison to the unvaccinated animals. At the end of the trial, a higher viral load was found in several lymphatic tissues and the ileum of unvaccinated pigs in comparison to vaccinated pigs. The difference between groups was especially apparent in the ileum. The results presented here demonstrate a possible use for recombinant S. cerevisiae expressing viral proteins as an oral vaccine against PCV2. A powdered freeze-dried recombinant S. cerevisiae used as an oral vaccine could be mixed with feed and may offer a cheap and less labour intensive alternative to inoculation with the additional advantage that no cooling chain would be required for vaccine transport and storage.

  2. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    Science.gov (United States)

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  3. Expression of Major Capsid Protein of Cainine Parvovirus by Yeast (Pichia pastoris and Efficient Purification using Arginine in Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Ying He

    2016-06-01

    Full Text Available An immunochromatographic (IC assay was developed for rapid detection of canine parvovirus using the monoclonal antibodies (McAbs against canine parvovirus (CPV-2. To prepare the McAbs, gene encoding the VP2 protein of CPV-2a was expressed in a Pichia pastoris expression vector pPICZ-A. The recombinant VP2 was similar antigenically function to the native capsid protein as demonstrated by Western blotting using CPV- 2 polyclonal antiserum. McAbs against CPV-2 were produced by fusing myeloma cell line SP2/0 with spleen cells from Balb/C mice immunized with purified recombinant VP2 protein. By ELISA it was shown that the McAbs specifically recognized VP2 epitopes of CPV-2 but not those of other canine viruses such as Canine distemper virus (CDV or canine adenovirus (CAV. An IC assay developed with the McAbs was suitable for rapid detection of canine parvovirus. Fecal samples (120 from dogs suspected of CPV-2 infection were analyzed by both haemaglutination (HA assay and the IC assay, and 52 and 53 samples were found positive for CPV-2, respectively. Comparison between the two different assays revealed that IC assay is as sensitive as HA; the sensitivity and specificity for the IC assay is 98.6% and 98.1%, respectively.

  4. Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

    Institute of Scientific and Technical Information of China (English)

    张友尚; 胡红明; 蔡若蓉; 冯佑民; 朱尚权; 贺潜斌; 唐月华; 徐明华; 许英镐; 张新堂; 刘滨; 梁镇和

    1996-01-01

    A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3’-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.

  5. Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco.

    Science.gov (United States)

    Liu, Ge-Yu; Zhang, Yu-Xiu; Chai, Tuan-Yao

    2011-06-01

    Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl(2) over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants. PMID:21327392

  6. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  7. Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Yadav, Shailesh Kumar R; Sahu, Tejram; Dixit, Aparna

    2016-08-01

    Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source. PMID:27020281

  8. Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis.

    Science.gov (United States)

    Takahashi, Shunsuke; Ando, Akira; Takagi, Hiroshi; Shima, Jun

    2009-11-01

    Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. PMID:19749072

  9. Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains

    DEFF Research Database (Denmark)

    Greppi, Anna; Krych, Lukasz; Costantini, Antonella;

    2015-01-01

    Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces...

  10. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular...... Compartments and Transport. Mating Type Switch. Molecular Genetics and Recombinant DNA....

  11. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11 and MDR3 (ABCB4 expressed in the yeast Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Philipp Ellinger

    Full Text Available The human liver ATP-binding cassette (ABC transporters bile salt export pump (BSEP/ABCB11 and the multidrug resistance protein 3 (MDR3/ABCB4 fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

  12. Global expression studies in baker's yeast reveal target genes for the improvement of industrially-relevant traits: the cases of CAF16 and ORC2

    Directory of Open Access Journals (Sweden)

    Randez-Gil Francisca

    2010-07-01

    Full Text Available Abstract Background Recent years have seen a huge growth in the market of industrial yeasts with the need for strains affording better performance or to be used in new applications. Stress tolerance of commercial Saccharomyces cerevisiae yeasts is, without doubt, a trait that needs improving. Such trait is, however, complex, and therefore only in-depth knowledge of their biochemical, physiological and genetic principles can help us to define improvement strategies and to identify the key factors for strain selection. Results We have determined the transcriptional response of commercial baker's yeast cells to both high-sucrose and lean dough by using DNA macroarrays and liquid dough (LD model system. Cells from compressed yeast blocks display a reciprocal transcription program to that commonly reported for laboratory strains exposed to osmotic stress. This discrepancy likely reflects differences in strain background and/or experimental design. Quite remarkably, we also found that the transcriptional response of starved baker's yeast cells was qualitatively similar in the presence or absence of sucrose in the LD. Nevertheless, there was a set of differentially regulated genes, which might be relevant for cells to adapt to high osmolarity. Consistent with this, overexpression of CAF16 or ORC2, two transcriptional factor-encoding genes included in this group, had positive effects on leavening activity of baker's yeast. Moreover, these effects were more pronounced during freezing and frozen storage of high-sucrose LD. Conclusions Engineering of differentially regulated genes opens the possibility to improve the physiological behavior of baker's yeast cells under stress conditions like those encountered in downstream applications.

  13. Cloning and Expression of Maltase Encoding Gene from Baker's Yeast and Optimization of the Expression Conditions%面包酵母麦芽糖酶的异源表达及产酶条件优化

    Institute of Scientific and Technical Information of China (English)

    宋海岩; 林雪; 柏晓雯; 张翠英; 肖冬光

    2015-01-01

    通过设计特异性引物从快速发酵面包酵母BY-14中PCR克隆获得麦芽糖酶编码基因。选择BamH I和Hind III分别为上下游引物的酶切位点,将麦芽糖酶基因克隆到pET28a载体上。表达质粒经PCR扩增、双酶切和测序进行验证。正确的表达质粒pET28a-Mal62导入大肠杆菌宿主BL(21),经IPTG诱导表达,结果显示,重组蛋白能够可溶性表达。随后对大肠杆菌产酶条件进行优化,结果表明,当IPTG终浓度为0.4 mmol/L,菌浓OD600达到1.0时加入诱导剂,在26℃下振荡培养12 h可获得最高的产酶量39.9 U/L,比优化前提高了4倍。%The maltase encoding gene was amplified via PCR from baker’s yeast. Then it was digested by BamH I and Hind III, and inserted in-to plasmid pET28a. The expression plasmid was identified via PCR, restriction endonuclease digestion and sequencing. The valid expression plasmid (pET28a-Mal62) was transformed into competence Escherichia coli BL(21) for heterologous expression. The experimental results showed that, the target protein was able to be expressed and most of them were soluble. Afterwards, the expression conditions for the target pro-tein was optimized as follows:as IPTG concentration was 0.4 mmol/L, the induced timing OD600 for 1.0, oscillation induced culture for 12 h at 26℃, it was feasible to express the recombinant fusion protein in the supernatant with the highest production of 39.9 U/L, which increased by 4 fold compared with unoptimized conditions.

  14. Yeast cell factories on the horizon

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2015-01-01

    For thousands of years, yeast has been used for making beer, bread, and wine. In modern times, it has become a commercial workhorse for producing fuels, chemicals, and pharmaceuticals such as insulin, human serum albumin, and vaccines against hepatitis virus and human papillomavirus. Yeast has also...... been engineered to make chemicals at industrial scale (e.g., succinic acid, lactic acid, resveratrol) and advanced biofuels (e.g., isobutanol) (1). On page 1095 of this issue, Galanie et al. (2) demonstrate that yeast can now be engineered to produce opioids (2), a major class of compounds used...... for treating severe pain. Their study represents a tour de force in the metabolic engineering of yeast, as it involved the expression of genes for more than 20 enzymatic activities from plants, mammals, bacteria, and yeast itself. It clearly represents a breakthrough advance for making complex natural products...

  15. "Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

    Science.gov (United States)

    Sinha, Indu; Karagoz, Kubra; Fogle, Rachel L; Hollenbeak, Christopher S; Zea, Arnold H; Arga, Kazim Y; Stanley, Anne E; Hawkes, Wayne C; Sinha, Raghu

    2016-04-01

    Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance. PMID:27027327

  16. Expression patterns of the rice class I metallothionein gene family in response to lead stress in rice seedlings and functional complementation of its members in lead-sensitive yeast cells

    Institute of Scientific and Technical Information of China (English)

    XU YuFeng; ZHOU GongKe; ZHOU Lu; LI YiQin; LIU JinYuan

    2007-01-01

    Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions.In this paper, Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings.With the exception of OsMT-I-3b, the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots.Of these genes, transcription of OsMT-I-1a, OsMT-I-1b, OsMT-I-2c, OsMT-I-4a, OsMT-I-4b and OsMT-I-4c increased significantly, while transcription of OsMT-I-2a and OsMT-I-3a increased marginally.In contrast, the expression of OsMT-I-2b was inhibited.Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots, but had no obvious effects on the expression of OsMT-I-1a, OsMT-I-1b, OsMT-I-4a and OsMT-I-4b.All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells.These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate increased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is.This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress.

  17. Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress

    NARCIS (Netherlands)

    Gostincar, C.; Turk, M.; Trbuha, T.; Vaupotic, T.; Plemenitas, A.; Gunde-Cimerman, N.

    2008-01-01

    Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modi

  18. Fully automated molecular biology routines on a plasmid-based functional proteomic workcell: Evaluation and Characterization of Yeast Strains Optimized for Growth on Xylose Expressing "Stealth" Insecticidal Peptides.

    Science.gov (United States)

    Optimization of genes important to production of fuel ethanol from hemicellulosic biomass for use in developing improved commercial yeast strains is necessary to meet the rapidly expanding need for ethanol. The United States Department of Agriculture has developed a fully automated platform for mol...

  19. Yeast Interacting Proteins Database: YFR015C, YJL137C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...pression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into stationary

  20. Accelerating Yeast Prion Biology using Droplet Microfluidics

    Science.gov (United States)

    Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

    2012-02-01

    Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

  1. Vaginal yeast infection

    Science.gov (United States)

    Yeast infection - vagina; Vaginal candidiasis; Monilial vaginitis ... Most women have a vaginal yeast infection at some time. Candida albicans is a common type of fungus. It is often found in small amounts in the vagina , ...

  2. Yeast Infection (Candidiasis)

    Science.gov (United States)

    ... rash and rashes clinical tools newsletter | contact Share | Yeast Infection (Candidiasis) Information for adults A A A This is a candida (yeast) infection of the skin folds of the abdomen. Overview ...

  3. Prions in Yeast

    OpenAIRE

    Liebman, Susan W; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in unde...

  4. Combinatorial pathway assembly in yeast

    Directory of Open Access Journals (Sweden)

    Khalil Essani

    2015-10-01

    Full Text Available With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement whole natural or synthetic pathways into microorganisms. For this purpose balanced enzyme activities throughout the pathway need to be achieved in addition to simple functional gene expression to avoid bottlenecks and to obtain high titers of the desired product. As the optimization of pathways in a specific biological context is often hard to achieve by rational design, combinatorial approaches have been developed to address this issue. Here, current strategies and proof of concepts for combinatorial pathway assembly in yeasts are reviewed. By exploiting its ability to join multiple DNA fragments in a very efficient and easy manner, the yeast Saccharomyces cerevisiae does not only constitute an attractive host for heterologous pathway expression, but also for assembling pathways by recombination in vivo.

  5. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    Science.gov (United States)

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  6. Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

    OpenAIRE

    Southgate, V J; Steyn, A J; Pretorius, I. S.; van Vuuren, H J

    1993-01-01

    Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

  7. Biosensor analyzer for BOD index express control on the basis of the yeast microorganisms Candida maltosa, Candida blankii, and Debaryomyces hansenii.

    Science.gov (United States)

    Arlyapov, Viacheslav; Kamanin, Stanislav; Ponamoreva, Olga; Reshetilov, Anatoly

    2012-04-01

    The parameters of biosensors based on the yeast strains Candida maltosa VKM Y-2359, Candida blankii VKM Y-2675, and Debaryomyces hansenii VKM Y-2482 for biochemical oxygen demand (BOD) detection are compared. The catalytic activity of the strains was analyzed in relation to the growth phase. The possibility of using D. hansenii as a basis for receptor element of a biosensor for BOD detection in municipal and biotechnological wastewaters was shown.

  8. 游离高表达Mal62基因对面包酵母耐冷冻性的影响%Effects of High-expressed Ma162 Gene on Freezing Tolerance of Baker's Yeast

    Institute of Scientific and Technical Information of China (English)

    孙溪; 张翠英; 董建; 吴鸣月; 王光路; 肖冬光

    2012-01-01

    通过测定胞内海藻糖积累量、冷冻前后相对发酵力以及存活率的变化,对比游离高表达麦芽糖酶基因(Md62)的突变株BYCPM与亲本BY14的海藻糖合成能力,研究Ma/62基因游离高表达与酵母耐冷冻性之间的关系。结果表明.Ma/62基因游离高表达与酵母耐冷冻性有一定的相关性,突变株耐冷冻性改善,其在烘焙产业中具有潜在商业价值。%AThe relations between high-expressed Ma162 gene and freezing tolerance of baker's yeast were investigated through measuring the accumulating quantity of intracellular trehalose, observing the change of cell fermenting power and cell viability before and after freezing, and com- paring trehalose synthesis of parent strain BY14 and mutant strain BYCPM. The results showed that there was certain relativity between high-expressed ma/62 gene and freezing tolerance of baker' s yeast (freezing tolerance got improved for mutant strain). Accordingly, the improved freezing tolerance of BYCPM may make it useful in commercial baking industry.

  9. Molecular cloning and nucleotide sequence of the 1,2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells.

    Science.gov (United States)

    Inoue, T; Yoshida, T; Ichishima, E

    1995-12-01

    A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0. PMID:8519794

  10. Expression of LIP1 and LIP2 genes from Geotrichum species in Baker's yeast strains and their application to the bread-making process.

    Science.gov (United States)

    Monfort, A; Blasco, A; Sanz, P; Prieto, J A

    1999-02-01

    Lipolytic baker's yeast strains able to produce extracellular active lipase have been constructed by transformation with plasmids containing the LIP1 and LIP2 genes from Geotrichum sp. under the control of the Saccharomyces cerevisiae actin promoter (pACT1). Lipase productivity differed between both constructs, YEpACT-LIP1-t and YEpACT-LIP2-t, being higher for the strain bearing the LIP2 gene in all culture media tested. This result appeared not to be the consequence of a defect in the transcription of the LIP1 gene as revealed by Northern blot analysis. Replacing the signal sequence of LIP1 by that of LIP2 in the YEpACT-LIP1-t plasmid enhanced significantly the secretion of lipase 1, but the levels of lipase activity were still lower than those found for the YEpACT-LIP2-t transformant. Recombinant lipase 2 protein produced by baker's yeast exhibited biochemical properties similar to those of the natural enzyme. Fermented dough prepared with YEpACT-LIP2-t-carrying cells rendered a bread with a higher loaf volume and a more uniform crumb structure than that prepared with control yeast. These effects were stronger by the addition in the bread dough formulas of a preferment enriched in recombinant lipase 2.

  11. Yeast That Smell

    Directory of Open Access Journals (Sweden)

    Eugenia Y Xu

    2008-08-01

    Full Text Available The fundamental mechanism of olfactory receptor activation has been conserved from yeast to humans. Engineered yeast cells can smell some of the same odorants as humans can, which makes yeast an ideal model system for studying human olfaction. Furthermore, if engineered yeast cells are incorporated into sensory arrays, they can be used as biosensors or artificial noses.Keywords: Yeast, olfactory receptor, G protein-coupled receptor, biosensor, smellReceived: 31 July 2008 / Received in revised form: 6 August 2008, Accepted: 13 August 2008, Published online: 17 August 2008

  12. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    Science.gov (United States)

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  13. Ranking the European armed forces

    NARCIS (Netherlands)

    Beeres, R.J.M.; Bogers, M.

    2012-01-01

    The degree of collectiveness aimed for in European defence policy raises issues such as burden sharing and relative performance measurement of the European Armed Forces (EAF). This paper compares EAF performance rates on three dimensions: input, throughput and output. In order to express performance

  14. 人源蛋白酶体α亚基6的酵母表面展示及表达优化%YEAST SURFACE DISPLAY OF HUMAN PROTEASOME SUBUNIT ALPHA 6 AND EXPRESSION ENHANCEMENT

    Institute of Scientific and Technical Information of China (English)

    唐语谦; 林影; 韩双艳; 梁世中

    2007-01-01

    为构建人源蛋白酶体α亚基6(hPSA6)的酵母表面展示体系用于抗体表位分析和研究泛素-蛋白酶体途径,并优化hPSA6的展示表达,将基因PSA6_HUMAN克隆于表达载体pICAS-H,该载体带有His.tag标签可检测表达水平.经过重组酵母的培养,用抗His.tag或抗hPSA6的单克隆抗体和荧光二抗进行免疫荧光染色,通过流式细胞仪和荧光显微镜检测到hPSA6已经成功地展示于酿酒酵母表面.通过对培养基中不同初始浓度的葡萄糖和酸水解酪素的诱导培养,发现hPSA6呈现出不同的展示水平,与对照相比,对His.tag进行免疫荧光检测观测到3%酸水解酪素诱导培养可获得超过70%的展示表达率,3%葡萄糖诱导培养可达到50%以上表达率,2%葡萄糖诱导也有接近40%表达率.考虑到葡萄糖效应和灭菌过程中高浓度的葡萄糖易碳化,采用2%葡萄糖进行诱导表达较3%更适合于hPSA6的展示表达.%To construct the yeast display system of the human proteasome subunit alpha 6(hPSA6) for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway,and enhance the display expression of hPSA6, the gene PSA6_HUMAN coding hPSA6 was cloned into a yeast-displaying expression vector, pICAS-H, which had been inserted a His. Tag marker for expression level detection. As probed with a his. Tag monoclonal antibody (Mab) and corresponding Mab, hPSA6 was detected functionally by flow cytometry and fluorescence microscopy analysis which confirmed that yeast-displaying recombinant hPSA6 with highly specific affinity was expressed efficiently after 24 h cultivation of recombinant yeast MT8-1/pICAS-H-PSA6. Induced by different concentration of initial glucose and casein acid in restrictive mediums, the displayed hPSA6 expressions were compared through immunofluorescence by using anti-His Mabs. Comparing with negative control, more than 70% cells were induced to express hPSA6 in SA with 3% casein acid initially. The 3

  15. Overexpression of membrane proteins from higher eukaryotes in yeasts.

    Science.gov (United States)

    Emmerstorfer, Anita; Wriessnegger, Tamara; Hirz, Melanie; Pichler, Harald

    2014-09-01

    Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals. PMID:25070595

  16. [Expression of SM30 (A-F) Genes Encoding Spicule Matrix Proteins in Intact and Damaged Sea Urchin Strongylocentrotus intermedius (A. Agassiz, 1863) at the Six-Arm Pluteus].

    Science.gov (United States)

    Sharmankina, V V; Kiselev, K V

    2016-03-01

    In this study we investigated expression of the SM30(A-F) gene family encoding Strongylocentrotus intermedius spicule matrix proteins during the normal and regenerative pluteus II stage (three pairs of arms). We found that SiSM30A and SiSM30B genes are expressed at high levels in the normal pluteus II sea urchin. SiSM30A is expression was also significantly upregulated in the reparative pluteus II stage 3 hours after damage. Conversely, SiSM30B was downregulated during the reparative pluteus II stage. Our findings reveal a substantial similarity between the activity of SiSM30A and SiSM30B activity in the processes of regenerative growth during the pluteus II stage and during normal development at the prism stage in Strongylocentrotus purpuratus. On the basis of our findings, we propose that normal developmental mechanisms corresponding to the preceding developmental stage are reactivated during pluteus regeneration.

  17. 脲基酰胺酶基因在黄酒酵母中的整合型表达%Constitutive expression of DUR1 ,2 gene in Chinese rice wine yeast

    Institute of Scientific and Technical Information of China (English)

    朱旭亚; 陆健; 谢广发

    2012-01-01

    Ethyl carbamate(EC) is a potential carcinogen for human, which can be found in many fermented alcoholic beverages.The main precursor for EC is urea produced by yeast.The DUR1,2 gene which encoded urea amidolyase was cloned into plasmid pYX212 between the TPI promoter and terminator sites to form the DUR! ,2 expression cassette.The cassette was then integrated into the genome of the Chinese rice wine yeast 85#.Thus a urea-degrading Chinese rice wine yeast 85 was obtained.According to the laboratory scale rice wine brewing tests,urea was reduced to 8.34mg/L by yeast 85 69.9% less than the parental strain, while the ethyl carbamate level was 40.5% less than that of the parental strain after a period of storage.Meanwhile,85 was substantially equivalent to its parental strain in terms of fermentation ability, ethanol ,total acid and amino nitrogen.%氨基甲酸乙酯是一种人类的潜在致癌物,在许多发酵酒中均有存在,主要来源于发酵过程中酵母代谢产生的尿素。以pYX212为载体,将脲基酰胺酶基因DURl,2克隆到TPI强启动子和终止子之间的位点,再通过同源重组的方式将受强启动子调控的目的基因整合到黄酒酵母的基因组中,最终获得一株低产尿素的胞内脲基酰胺酶基因组成型高表达的黄酒酵母85在实验室规模的黄酒酿造实验中,85产尿素量为8.34mg/L,比出发菌株降低了69.9%,贮存一段时间后的酒液中氨基甲酸乙酯含量比出发菌株降低了40.5%,而发酵性能、酒精度、总酸及氨基态氮与出发菌株无显著差异,

  18. Mechanisms of expression and translocation of major fission yeast glucose transporters regulated by CaMKK/phosphatases, nuclear shuttling, and TOR.

    Science.gov (United States)

    Saitoh, Shigeaki; Mori, Ayaka; Uehara, Lisa; Masuda, Fumie; Soejima, Saeko; Yanagida, Mitsuhiro

    2015-01-15

    Hexose transporters are required for cellular glucose uptake; thus they play a pivotal role in glucose homeostasis in multicellular organisms. Using fission yeast, we explored hexose transporter regulation in response to extracellular glucose concentrations. The high-affinity transporter Ght5 is regulated with regard to transcription and localization, much like the human GLUT transporters, which are implicated in diabetes. When restricted to a glucose concentration equivalent to that of human blood, the fission yeast transcriptional regulator Scr1, which represses Ght5 transcription in the presence of high glucose, is displaced from the nucleus. Its displacement is dependent on Ca(2+)/calmodulin-dependent kinase kinase, Ssp1, and Sds23 inhibition of PP2A/PP6-like protein phosphatases. Newly synthesized Ght5 locates preferentially at the cell tips with the aid of the target of rapamycin (TOR) complex 2 signaling. These results clarify the evolutionarily conserved molecular mechanisms underlying glucose homeostasis, which are essential for preventing hyperglycemia in humans.

  19. Enhanced expression of the Erns protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals.

    Science.gov (United States)

    Luo, Yuzi; Li, Lin; Austermann-Busch, Sophia; Dong, Mei; Xu, Jingjing; Shao, Lina; Lei, Jianlin; Li, Na; He, Wen-Rui; Zhao, Bibo; Li, Su; Li, Yongfeng; Liu, Lihong; Becher, Paul; Sun, Yuan; Qiu, Hua-Ji

    2015-09-15

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF. PMID:26005003

  20. 酵母双杂交诱饵载体pGBKT7_MYC2构建及表达鉴定%Construction and Expressional Identification of Yeast Two - hybrid Bait Expression Vector pGBKT7_MYC2

    Institute of Scientific and Technical Information of China (English)

    刘武; 肖牧; 阮颖; 刘春林

    2012-01-01

    MYC2是一类含有helix - loop - helix (bHLH)结构域的转录因子.为进一步研究MYC2因子在植物防御抗性中的作用及其参与植物JA,SA等信号途径的作用机制,克隆了拟南芥的MYC2基因,以此构建了pGBKT7_MYC2酵母双杂交载体,通过Western blotting验证表明,该载体能在酵母细胞里正常表达.%MYC2, a basic helix - loop - helix (bHLH) domain - containing TF, acts as a positive regulator of abseisic acid - dependent drought responses and is also induced in JA - mediated responses. Taking the cDNA from Arabidophsis as the template, full - length COS of MYC2 had been cloned and then was ligated into the bait expression vector pG-BKT7. After verified by digestion, the bait vector was transformed into Clod yeast cells, and the expression of MYC2 gene was checked by Western blotting. As a result, the bait expression vector pGBKT7_MYC2 was constructed successfully, which laid the foundation for screening target proteins interacting and mapping the network with the MYC2 protein.

  1. Yeast systems for the commercial production of heterologous proteins.

    Science.gov (United States)

    Buckholz, R G; Gleeson, M A

    1991-11-01

    Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.

  2. Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

    OpenAIRE

    Elledge, S J; Mulligan, J T; Ramer, S W; Spottswood, M; Davis, R W

    1991-01-01

    This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system,...

  3. Structural Studies of the Yeast Mitochondrial Degradosome

    DEFF Research Database (Denmark)

    Feddersen, Ane; Jonstrup, Anette Thyssen; Brodersen, Ditlev Egeskov

    and nuclear exosome complexes, which consist of 10-12 different nuclease subunits, the mitochondrial degradosome is composed of only two large subunits - an RNase (Dss1p) and a helicase (Suv3p), belonging the Ski2 class of DExH box RNA helicases. Both subunits are encoded on the yeast nuclear genome...... and and Suv3p from the fission yeast, Schizosaccharomyces pombe, have been cloned for heterologous expression in E. coli. Of the two, we have succeeded in purifying the 73kDa Suv3p by Ni2+-affinity chromatography followed by cleavage of the N-terminal His-tag, cation exchange, and gel filtration. Crystals...

  4. The arms race control

    International Nuclear Information System (INIS)

    Written in 1961, this paper presents the content of a book entitled 'The arms race control' where the author outlined the difference between disarmament and arms control, described the economic and moral role of arms race, the importance of force balance for international security. He wandered whether arms control could ensure this balance and whether nuclear balance meant force balance. Force balance then appears to be a precarious and unsteady component of international security. He commented the challenges of disarmament, recalled some arguments for a nuclear disarmament. Then he discussed what would be an arms control with or without disarmament (either nuclear or conventional)

  5. Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.

    Science.gov (United States)

    Radovanovic, Natasa; Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Cloutier, Sylvie

    2014-07-01

    With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed. PMID:24522837

  6. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

  7. Modeling brewers' yeast flocculation

    Science.gov (United States)

    van Hamersveld EH; van der Lans RG; Caulet; Luyben

    1998-02-01

    Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.

  8. Meiosis in haploid yeast

    OpenAIRE

    Wagstaff, Joseph E.; Klapholz, Sue; Esposito, Rochelle Easton

    1982-01-01

    Haploid yeast cells normally contain either the MATa or MATα mating-type allele and cannot undergo meiosis and spore formation. If both mating-type alleles are present as a consequence of chromosome III disomy (MATa/MATα), haploids initiate meiosis but do not successfully form spores, probably because the haploid chromosome complement is irregularly partitioned during meiotic nuclear division. We have demonstrated that the ochre-suppressible mutation spo13-1 enables haploid yeast cells disomi...

  9. Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation

    DEFF Research Database (Denmark)

    Graakjaer, Jesper; Christensen, Rikke; Kolvraa, Steen;

    2007-01-01

    BACKGROUND: Previous studies have demonstrated that telomeres in somatic cells are not randomly distributed at the end of the chromosomes. We hypothesize that these chromosome arm specific differences in telomere length (the telomere length pattern) may be actively maintained. In this study we...... investigate the existence and maintenance of the telomere length pattern in stem cells. For this aim we studied telomere length in primary human mesenchymal stem cells (hMSC) and their telomerase-immortalised counterpart (hMSC-telo1) during extended proliferation as well as after irradiation. Telomere lengths...... were measured using Fluorescence In Situ Hybridization (Q-FISH). RESULTS: A telomere length pattern was found to exist in primary hMSC's as well as in hMSC-telo1. This pattern is similar to what was previously found in lymphocytes and fibroblasts. The cells were then exposed to a high dose of ionizing...

  10. Yeast flocculation: what brewers should know.

    Science.gov (United States)

    Verstrepen, K J; Derdelinckx, G; Verachtert, H; Delvaux, F R

    2003-05-01

    For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.

  11. Production of alpha-amylase by yeast

    Energy Technology Data Exchange (ETDEWEB)

    Thomse, K.K.

    1987-01-01

    The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experiments will be discussed. (Refs. 21).

  12. The UDP-glycosyltransferase (UGT) superfamily expressed in humans, insects and plants: Animal-plant arms-race and co-evolution.

    Science.gov (United States)

    Bock, Karl Walter

    2016-01-01

    UDP-glycosyltransferases (UGTs) are major phase II enzymes of a detoxification system evolved in all kingdoms of life. Lipophilic endobiotics such as hormones and xenobiotics including phytoalexins and drugs are conjugated by vertebrates mainly with glucuronic acid, by invertebrates and plants mainly with glucose. Plant-herbivore arms-race has been the major driving force for evolution of large UGT and other enzyme superfamilies. The UGT superfamily is defined by a common protein structure and signature sequence of 44 amino acids responsible for binding the UDP moiety of the sugar donor. Plants developed toxic phytoalexins stored as glucosides. Upon herbivore attack these conjugates are converted to highly reactive compounds. In turn, animals developed large families of UGTs in their intestine and liver to detoxify these phytoalexins. Interestingly, phytoalexins, exemplified by quercetin glucuronides and glucosinolate-derived isocyanates, are known insect attractant pigments in plants, and antioxidants, anti-inflammatory and chemopreventive compounds of humans. It is to be anticipated that phytochemicals may provide a rich source in beneficial drugs.

  13. HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

    OpenAIRE

    Hall, Caitlin; Nelson, David M.; Ye, Xiaofen; Baker, Kayla; DeCaprio, James A.; Seeholzer, Steven; Lipinski, Marc; Adams, Peter D

    2001-01-01

    Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo s...

  14. 石斑鱼β-防御素的酵母表达及其产物抗菌活性分析%THE YEAST EXPRESSION OF GROUPER β-DEFENSIN AND ANTIBACTERIAL ACTIVITY OF THE RECOMBINANT PROTEIN

    Institute of Scientific and Technical Information of China (English)

    金俊琰; 周莉; 桂建芳

    2011-01-01

    防御素是一类阳离子抗菌肽.研究从石斑鱼垂体SMART cDNA文库中扩增出129 bp石斑鱼β-防御素成熟肽序列,将其克隆到毕赤酵母表达载体pPCIZαA中,构建了石斑鱼β-防御素的真核表达载体,电击转化毕赤酵母GS115.Western Blot分析表明石斑鱼β-防御素在酵母菌中获得了表达.体外抗菌实验表明纯化的重组蛋白具有抑制大肠杆菌以及嗜水气单胞菌的作用,但是对革兰氏阳性菌,如金黄色葡萄球菌和藤黄微球菌的生长没有抑制作用.实验结果表明酵母表达的石斑鱼β-防御素能够特异地抑制革兰氏阴性菌的生长.%Defensin, a small cationic peptide, is an antimicrobial peptide, exhibit broad-spectrum antibacterial activity. They spread widely in plants, invertebrate and vertebrate animals, which can rapidly kill bacteria, fungi and viruses etc. Bacteria and viruses limited the production of grouper (Epinephelus coioides). In this study, we cloned grouper P-defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper P-defensin cDNA was amplified by polymerase chain reaction (PCR) from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper. The 129 bp DNA fragment encoding mature grouper P-defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPClZaA and transfected into Pichia pastoris GS115 expression host by electroporation. The genome of Pichia pastoris clone was extracted as template for screened the positive clone by using PCR. After grouper p-defensin was secreted by GS 115 by small-scale culture, the recombinant grouper P-defensin was induced by methanol for large scale. The supernatant was collected in 24h, 48h and 72h post induction for the recombinant protein detection. Subsequently, the grouper p-defensin peptide was detected in the supernatant of transfected yeast by Western Blot

  15. Effect of salt hyperosmotic stress on yeast cell viability

    Directory of Open Access Journals (Sweden)

    Logothetis Stelios

    2007-01-01

    Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

  16. Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

    Directory of Open Access Journals (Sweden)

    Reza Saberianfar

    Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

  17. A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

    DEFF Research Database (Denmark)

    Kampranis, S C; Damianova, R; Atallah, M;

    2000-01-01

    The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allo...

  18. Genetics of Yeasts

    Science.gov (United States)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  19. Selection of functional cDNAs by complementation in yeast.

    OpenAIRE

    McKnight, G L; McConaughy, B L

    1983-01-01

    Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the...

  20. Managing new arms races

    International Nuclear Information System (INIS)

    The management of new arms races in the region of Asia-Pacific includes considerations of weapons trade and transfer in the region, with an emphasis on nuclear weapons proliferation. It deals with the problem of controlling the arms trade and the efforts to control conventional weapons and underlines the possible role and influence of Conference on Cooperation and Security in Europe (CSCE)

  1. ARM Mentor Selection Process

    Energy Technology Data Exchange (ETDEWEB)

    Sisterson, D. L. [Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-10-01

    The Atmospheric Radiation Measurement (ARM) Program was created in 1989 with funding from the U.S. Department of Energy (DOE) to develop several highly instrumented ground stations to study cloud formation processes and their influence on radiative transfer. In 2003, the ARM Program became a national scientific user facility, known as the ARM Climate Research Facility. This scientific infrastructure provides for fixed sites, mobile facilities, an aerial facility, and a data archive available for use by scientists worldwide through the ARM Climate Research Facility—a scientific user facility. The ARM Climate Research Facility currently operates more than 300 instrument systems that provide ground-based observations of the atmospheric column. To keep ARM at the forefront of climate observations, the ARM infrastructure depends heavily on instrument scientists and engineers, also known as lead mentors. Lead mentors must have an excellent understanding of in situ and remote-sensing instrumentation theory and operation and have comprehensive knowledge of critical scale-dependent atmospheric processes. They must also possess the technical and analytical skills to develop new data retrievals that provide innovative approaches for creating research-quality data sets. The ARM Climate Research Facility is seeking the best overall qualified candidate who can fulfill lead mentor requirements in a timely manner.

  2. Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris Imunogenicidade e antigenicidade da proteína recombinante EMA-1 de Theileria equi expressa em Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Leandro Q. Nizoli

    2009-06-01

    Full Text Available The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg.L-1 of recombinant protein. The protein was concentrated and detected in Dot blot. The recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with T. equi. The immunogenicity of rEMA-1 protein was demonstrated by IFAT using sera from recombinant-protein-immunized mice using aluminum hydroxide as adjuvant. All animals vaccinated with rEMA-1 developed a high specific antibody response. This results suggest that rEMA-1expressed in P. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.A piroplasmose equina causada por Theileria equi é uma das mais importantes doenças parasitárias de equídeos, causando danos a saúde animal e perdas econômicas. Em T. equi, 2 proteínas de superfície de merozoítos, equi merozoite antigen EMA-1 e EMA-2, têm sido identificadas como antígenos imunodominantes. Sugerindo que estes antígenos possam ser usados como produtos imunobiológicos. O gene EMA-1 de T. equi foi clonado e expressado na levedura Pichia pastoris. As leveduras transformadas foram cultivadas a altas densidades celulares expressando 389 mg.L-1 de proteína recombinante. A proteína foi concentrada e detectada em Dot blot. O produto recombinante foi antigenicamente similar à proteína nativa quando determinado usando anticorpo

  3. Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

    Science.gov (United States)

    Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

  4. Opportunistic Pathogenic Yeasts

    Science.gov (United States)

    Banerjee, Uma

    Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as ‘opportunistic pathogenic yeasts.’ However, the term ‘pathogenic’ is quite tricky, as it depends of various factors of the host, the ‘bug’ and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 ° C (normal body temperature of human), can be pathogenic and cause infection in particular situation

  5. Nonspecific Arm Pain

    Directory of Open Access Journals (Sweden)

    Ali Moradi

    2013-12-01

    Full Text Available   Nonspecific activity-related arm pain is characterized by an absence of objective physical findings and symptoms that do not correspond with objective pathophysiology. Arm pain without strict diagnosis is often related to activity, work-related activity in particular, and is often seen in patients with physically demanding work. Psychological factors such as catastrophic thinking, symptoms of depression, and heightened illness concern determine a substantial percentage of the disability associated with puzzling hand and arm pains. Ergonomic modifications can help to control symptoms, but optimal health may require collaborative management incorporating psychosocial and psychological elements of illness.

  6. Nonspecific Arm Pain

    Directory of Open Access Journals (Sweden)

    Ali Moradi

    2013-12-01

    Full Text Available Nonspecific activity-related arm pain is characterized by an absence of objective physical findings and symptoms that do not correspond with objective pathophysiology. Arm pain without strict diagnosis is often related to activity, work-related activity in particular, and is often seen in patients with physically demanding work. Psychological factors such as catastrophic thinking, symptoms of depression, and heightened illness concern determine a substantial percentage of the disability associated with puzzling hand and arm pains. Ergonomic modifications can help to control symptoms, but optimal health may require collaborative management incorporating psychosocial and psychological elements of illness.

  7. An Elastica Arm Scale

    CERN Document Server

    Bosi, F; Corso, F Dal; Bigoni, D

    2015-01-01

    The concept of 'deformable arm scale' (completely different from a traditional rigid arm balance) is theoretically introduced and experimentally validated. The idea is not intuitive, but is the result of nonlinear equilibrium kinematics of rods inducing configurational forces, so that deflection of the arms becomes necessary for the equilibrium, which would be impossible for a rigid system. In particular, the rigid arms of usual scales are replaced by a flexible elastic lamina, free of sliding in a frictionless and inclined sliding sleeve, which can reach a unique equilibrium configuration when two vertical dead loads are applied. Prototypes realized to demonstrate the feasibility of the system show a high accuracy in the measure of load within a certain range of use. It is finally shown that the presented results are strongly related to snaking of confined beams, with implications on locomotion of serpents, plumbing, and smart oil drilling.

  8. ARM7-kehityskortti

    OpenAIRE

    Kukkonen, Henri

    2006-01-01

    Tämän opinnäytetyön tarkoituksena oli suunnitella ja toteuttaa ARM-mikro-ohjain pohjainen kehityskortti. Kortin tuli olla soveltuva ARM-ohjelmoinnin opettamiseen. Työssä myös selvitettiin ARM-mikro-ohjaimen ohjelmointiympäristön käyttöönotto. Teoriaosassa käsitellään ARM-arkkitehtuuria, työssä käytettyjen Atmelin AT91R40008-mikro-ohjaimen sekä Philipsin LPC2105-mikro-ohjaimen ominaisuuksia. Erityisesti työssä keskitytään kehityskorttien suunnitteluun. Kehityskortin vaatimuksina oli, että se ...

  9. Biofuels. Altered sterol composition renders yeast thermotolerant

    DEFF Research Database (Denmark)

    Caspeta, Luis; Chen, Yun; Ghiaci, Payam;

    2014-01-01

    adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol...... desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype....

  10. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference

    OpenAIRE

    Murphy, Katherine A.; Christine A. Tabuloc; Cervantes, Kevin R.; Joanna C. Chiu

    2016-01-01

    RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally...

  11. The use of high-selenium yeast to raise selenium status: how does it measure up?

    OpenAIRE

    Rayman, MP

    2004-01-01

    Selenium-enriched yeast (Se-yeast) is a common form of selenium used to supplement dietary intake of this important trace mineral. However, its availability within the EU is under threat owing to concerns expressed by the EC Scientific Committee on Food that Se-yeast supplements are poorly characterised and could potentially cause the build up of selenium in tissues to toxic levels. This review examines the validity of these concerns. Diagrams of the biosynthesis and metabolism of selenium co...

  12. Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV.

    OpenAIRE

    Popoff, S C; Spira, A I; Johnson, A. W.; Demple, B

    1990-01-01

    DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme. We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11. Gene disruption experiments confirm that the Apn1 protein accounts for greater...

  13. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    Science.gov (United States)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  14. Hello to Arms

    Science.gov (United States)

    2005-01-01

    This image highlights the hidden spiral arms (blue) that were discovered around the nearby galaxy NGC 4625 by the ultraviolet eyes of NASA's Galaxy Evolution Explorer. The image is composed of ultraviolet and visible-light data, from the Galaxy Evolution Explorer and the California Institute of Technology's Digitized Sky Survey, respectively. Near-ultraviolet light is colored green; far-ultraviolet light is colored blue; and optical light is colored red. As the image demonstrates, the lengthy spiral arms are nearly invisible when viewed in optical light while bright in ultraviolet. This is because they are bustling with hot, newborn stars that radiate primarily ultraviolet light. The youthful arms are also very long, stretching out to a distance four times the size of the galaxy's core. They are part of the largest ultraviolet galactic disk discovered so far. Located 31 million light-years away in the constellation Canes Venatici, NGC 4625 is the closest galaxy ever seen with such a young halo of arms. It is slightly smaller than our Milky Way, both in size and mass. However, the fact that this galaxy's disk is forming stars very actively suggests that it might evolve into a more massive and mature galaxy resembling our own. The armless companion galaxy seen below NGC 4625 is called NGC 4618. Astronomers do not know why it lacks arms but speculate that it may have triggered the development of arms in NGC 4625.

  15. Design of synthetic yeast promoters via tuning of nucleosome architecture.

    Science.gov (United States)

    Curran, Kathleen A; Crook, Nathan C; Karim, Ashty S; Gupta, Akash; Wagman, Allison M; Alper, Hal S

    2014-01-01

    Model-based design of biological parts is a critical goal of synthetic biology, especially for eukaryotes. Here we demonstrate that nucleosome architecture can have a role in defining yeast promoter activity and utilize a computationally-guided approach that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters. Initially, we use our approach to reprogram native promoters for increased expression and evaluate their performance in various genetic contexts. Increases in expression ranging from 1.5- to nearly 6-fold in a plasmid-based system and up to 16-fold in a genomic context were obtained. Next, we demonstrate that, in a single design cycle, it is possible to create functional, purely synthetic yeast promoters that achieve substantial expression levels (within the top sixth percentile among native yeast promoters). In doing so, this work establishes a unique DNA-level specification of promoter activity and demonstrates predictive design of synthetic parts. PMID:24862902

  16. Glutathione Production in Yeast

    Science.gov (United States)

    Bachhawat, Anand K.; Ganguli, Dwaipayan; Kaur, Jaspreet; Kasturia, Neha; Thakur, Anil; Kaur, Hardeep; Kumar, Akhilesh; Yadav, Amit

    Glutathione, γ -glutamyl-cysteinyl-glycine, is the most abundant non-protein thiol found in almost all eukaryotic cells (and in some prokaryotes). The tripeptide, which is synthesized non-ribosomally by the consecutive action of two soluble enzymes, is needed for carrying out numerous functions in the cell, most important of which is the maintenance of the redox buffer. The cycle of glutathione biosynthesis and degradation forms part of the γ -glutamyl cycle in most organisms although the latter half of the pathway has not been demonstrated in yeasts. Our current understanding of how glutathione levels are controlled at different levels in the cell is described. Several different routes and processes have been attempted to increase commercial production of glutathione using both yeast and bacteria. In this article we discuss the history of glutathione production in yeast. The current bottlenecks for increased glutathione production are presented based on our current understanding of the regulation of glutathione homeostasis, and possible strategies for overcoming these limitations for further enhancing and improving glutathione production are discussed

  17. Oleaginous yeasts from Ethiopia.

    Science.gov (United States)

    Jiru, Tamene Milkessa; Abate, Dawit; Kiggundu, Nicholas; Pohl, Carolina; Groenewald, Marizeth

    2016-12-01

    Oleaginous microorganisms can produce high amounts of oil (>20 % of their biomass) under suitable cultivation conditions. In this research work 200 samples were collected from soil, plant surfaces (leaves, flowers and fruits), waste oils from traditional oil milling houses and dairy products (cheese, milk and yoghurt) in Ethiopia. Three hundred and forty yeast colonies were isolated from these samples. By applying Sudan III staining tests, 18 strains were selected as possible oleaginous yeasts. The 18 strains were identified and characterized for their lipid production as a feedstock for biodiesel production in the future. They were identified using morphological and physiological methods as well as sequencing the 3'end of the small-subunit rRNA gene, the internal transcribed spacer regions (ITS; ITS 1, ITS 2 and the intervening 5.8S rRNA gene), and the D1/D2 domain of the 26S rRNA gene. The 18 yeasts were identified as Cutaneotrichosporon curvatus (syn, Cryptococcus curvatus) (PY39), Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) (SY89), Rhodotorula dairenensis (SY94) and Rhodotourula mucilaginosa (SY09, SY18, SY20, PY21, PY23, PY25, SY30, PY32, SY43, PY44, SY52, PY55, PY61, SY75 and PY86). Under nitrogen-limited cultivation conditions, R. mucilaginosa PY44 produced the highest biomass (15.10 ± 0.54 g/L), while R. mucilaginosa PY32 produced the lowest biomass (10.32 ± 0.18 g/L). The highest lipid yield of 6.87 ± 0.62 g/L and lipid content of 46.51 ± 0.70 % were attained by C. curvatus (syn, C. curvatus) PY39. On the other hand, R. mucilaginosa PY61 gave the lowest lipid yield (2.06 ± 0.52 g/L) and R. mucilaginosa SY52 gave the lowest lipid content of 16.99 ± 0.85 %. The results in this research work suggest that much more oleaginous yeasts can be isolated from Ethiopian environment. On the basis of their substantial lipid production abilities, the three oleaginous yeast strains PY39, SY89 and SY18 were selected and

  18. Sexual differentiation in fission yeast

    DEFF Research Database (Denmark)

    Egel, R; Nielsen, O; Weilguny, D;

    1990-01-01

    The regulation of sexual reproduction in yeast constitutes the highest level of differentiation observed in these unicellular organisms. The various ramifications of this system involve DNA rearrangement, transcriptional control, post-translational modification (such as protein phosphorylation......) and receptor/signal processing. A few basic similarities are common to both fission and budding yeasts. The wiring of the regulatory circuitry, however, varies considerably between these divergent yeast groups....

  19. Flavour-active wine yeasts

    OpenAIRE

    Cordente, Antonio G.; Curtin, Christopher D.; Varela, Cristian; Pretorius, Isak S.

    2012-01-01

    The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can infl...

  20. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  1. Phoenix Stretches its Arm

    Science.gov (United States)

    2008-01-01

    [figure removed for brevity, see original site] Click on image for animation The Phoenix spacecraft is scheduled to begin raising its robotic arm up and out of its stowed configuration on the third Martian day, or Sol 3 (May 28, 2008) of the mission. This artist's animation, based on engineering models, shows how Phoenix will accomplish this task. First, its wrist actuator will rotate, releasing its launch-restraint pin. Next, the forearm moves up, releasing the elbow launch-restraint pin. The elbow will then move up and over in small steps, a process referred to as 'staircasing.' This ensures that the arm's protective biobarrier wrap, now unpeeled and lying to the side of the arm, will not get in the way of the arm's deployment. The arm is scheduled to straighten all the way out on Sol 4 (May 29, 2008), after engineers have reviewed images and telemetry data from the spacecraft showing that the biobarrier material has been cleared. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  2. Fully Automated Molecular Biology Routines on a Plasmid-Based Functional Proteomic Workcell: Evaluation and Characterization of Yeast Strains Optimized for Growth on Xylose and Engineered to Express an Insecticidal Peptide

    Science.gov (United States)

    Optimization of genes important to production of fuel ethanol from hemicellulosic biomass for use in engineering improved commercial yeast strains is necessary to meet the United States' rapidly expanding need for ethanol. United States Department of Agriculture, Agricultural Research Service, Nati...

  3. V5抗原表位融合性人雄激素受体基因酵母载体的构建及表达%The Construction and the Expression of V5 Epitope Fused Human Androgen Receptor Vector in the Yeast Cell

    Institute of Scientific and Technical Information of China (English)

    杨晨; 罗方妮; 戴卫星; 李姗姗; 黄仁华; 谢阳梅; 薛飞玥; 李湘鸣

    2013-01-01

    建立重组基因酵母筛选雄性内分泌干扰物的关键是雄激素受体(AR)在酵母细胞中的表达.为此,本文用PCR方法从W303-1A酵母基因组扩增3-磷酸甘油醛脱氢酶(GPD)启动子,将其插入到pYestrp2载体的SwaⅠ和BamH Ⅰ酶切位点,将所构建成的载体命名为pGPD.将5 '和3 '端含有BamH Ⅰ和EcoR Ⅰ黏性末端Ⅴ5抗原表位DNA,插入到pGPD相应的酶切位点,构建成pGPDV5载体.用PCR方法从pcDNA3.1/AR扩增2723 bp全长的AR开放性阅读框(ORF),将其插入到pGPDV5载体,构建成与V5抗原表位相融合的酵母表达载体pGPDV5/AR,将其转入W303-1A酵母细胞.用Western blot检测该酵母细胞提取液中的AR与V5相融合的表达蛋白,一抗选用小鼠源性V5单克隆抗体(IgG2a),二抗选用偶联有辣根过氧化物酶(HRP)的羊抗小鼠的多克隆抗体(IgG).结果表明,与V5相融合的AR蛋白在酵母细胞中成功表达.%When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors,the key procedure is the androgen receptor (AR) expression in the yeast cell.For this purpose,we obtained the GPD (glyceraldehyde-3-phosphme dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa Ⅰ and BamH Ⅰ sites of pYestrp2.The new constructed vector was named pGPD.The V5 epitope tag DNA with a 5'-BamH Ⅰ and a 3'-EcoR Ⅰ sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5.The 2723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR.This fused vector was transformed into the yeast cell (W303-1A).Western blot was used to detect the V5 fused protein of AR,in the protocol of which the primary monoclonal antibody (IgG2a) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the

  4. Arm To Arm Interface Using Embedded C

    Directory of Open Access Journals (Sweden)

    Mohanraj.C

    2013-02-01

    Full Text Available Embedded systems are the most emerging field in these recent years. In this paper a different number of ARM processors (LPC2148 and LPC2378 are interconnected using C for distributed services. N numbers of processors are connected as the network and each processing devices are interlinked with each other, so that the each data that is processed by the devices and it can be used by the other device to activate their entire process. All the processed data’s are communicated to other device through Xbee interface card. LPC2148 and LPC2378 ARM processors are used in this prototype and winXtalk is used as a software terminal window. In this paper, the ultimate benefits of multiple processor interactions related to the embedded applications and design issues of processor interconnection are discussed. The features of multiple processor interaction in inter process communication and executions of embedded multitasking are also discussed. In modern embedded computing platform, embedded processor used in various applications like home automation, industrial control, medical system, access control, etc. In this paper, using embedded processor interactions, the several data communication is established.

  5. Children In Armed Conflicts

    OpenAIRE

    Ranjan, Tejaswini

    2013-01-01

    This is a descriptive study. A child soldier is a child under the age of 18 that is recruited into the armed forces and engages in political violence. Child Soldiers are recruited by a state or non-state armed group and used as fighters, cooks, suicide bombers, human shields, messengers, spies, or for sexual purposes. This work of research describes the plight of child soldiers taking in context the scenario of different nations. The International mechanisms to combat this problem have also b...

  6. PHENIX Muon Arms

    International Nuclear Information System (INIS)

    The PHENIX Muon Arms detect muons at rapidities of |y|=(1.2-2.4) with full azimuthal acceptance. Each muon arm must track and identify muons and provide good rejection of pions and kaons (∼10-3). In order to accomplish this we employ a radial field magnetic spectrometer with precision tracking (Muon Tracker) followed by a stack of absorber/low resolution tracking layers (Muon Identifier). The design, construction, testing and expected run parameters of both the muon tracker and the muon identifier are described

  7. PHENIX Muon Arms

    Energy Technology Data Exchange (ETDEWEB)

    Akikawa, H.; Al-Jamel, A.; Archuleta, J.B.; Archuleta, J.R.; Armendariz, R.; Armijo, V.; Awes, T.C.; Baldisseri, A.; Barker, A.B.; Barnes, P.D.; Bassalleck, B.; Batsouli, S.; Behrendt, J.; Bellaiche, F.G.; Bland, A.W.; Bobrek, M.; Boissevain, J.G.; Borel, H.; Brooks, M.L.; Brown, A.W.; Brown, D.S.; Bruner, N.; Cafferty, M.M.; Carey, T.A.; Chai, J.-S.; Chavez, L.L.; Chollet, S.; Choudhury, R.K.; Chung, M.S.; Cianciolo, V.; Clark, D.J.; Cobigo, Y.; Dabrowski, C.M.; Debraine, A.; DeMoss, J.; Dinesh, B.V.; Drachenberg, J.L.; Drapier, O.; Echave, M.A.; Efremenko, Y.V.; En' yo, H.; Fields, D.E.; Fleuret, F.; Fried, J.; Fujisawa, E.; Funahashi, H.; Gadrat, S.; Gastaldi, F.; Gee, T.F.; Glenn, A.; Gogiberidze, G.; Gonin, M.; Gosset, J.; Goto, Y.; Granier de Cassagnac, R.; Hance, R.H.; Hart, G.W.; Hayashi, N.; Held, S.; Hicks, J.S.; Hill, J.C.; Hoade, R.; Hong, B.; Hoover, A.; Horaguchi, T.; Hunter, C.T.; Hurst, D.E.; Ichihara, T.; Imai, K.; Isenhower, L.D.L. Davis; Isenhower, L.D.L. Donald; Ishihara, M.; Jang, W.Y.; Johnson, J.; Jouan, D.; Kamihara, N.; Kamyshkov, Y.; Kang, J.H.; Kapoor, S.S.; Kim, D.J.; Kim, D.-W.; Kim, G.-B.; Kinnison, W.W.; Klinksiek, S.; Kluberg, L.; Kobayashi, H.; Koehler, D.; Kotchenda, L.; Kuberg, C.H.; Kurita, K.; Kweon, M.J.; Kwon, Y.; Kyle, G.S.; LaBounty, J.J.; Lajoie, J.G.; Lee, D.M.; Lee, S.; Leitch, M.J.; Li, Z.; Liu, M.X.; Liu, X.; Liu, Y.; Lockner, E.; Lopez, J.D.; Mao, Y.; Martinez, X.B.; McCain, M.C.; McGaughey, P.L.; Mioduszewski, S.; Mischke, R.E.; Mohanty, A.K.; Montoya, B.C.; Moss, J.M.; Murata, J.; Murray, M.M.; Nagle, J.L.; Nakada, Y.; Newby, J.; Obenshain, F.; Palounek, A.P.T.; Papavassiliou, V.; Pate, S.F.; Plasil, F.; Pope, K.; Qualls, J.M.; Rao, G.; Read, K.F. E-mail: readkf@ornl.gov; Robinson, S.H.; Roche, G.; Romana, A.; Rosnet, P.; Roth, R.; Saito, N.; Sakuma, T.; Sandhoff, W.F.; Sanfratello, L.; Sato, H.D.; Savino, R.; Sekimoto, M.; Shaw, M.R.; Shibata, T.-A.; Sim, K.S.; Skank, H.D.; Smith, D.E.; Smith, G.D. [and others

    2003-03-01

    The PHENIX Muon Arms detect muons at rapidities of |y|=(1.2-2.4) with full azimuthal acceptance. Each muon arm must track and identify muons and provide good rejection of pions and kaons ({approx}10{sup -3}). In order to accomplish this we employ a radial field magnetic spectrometer with precision tracking (Muon Tracker) followed by a stack of absorber/low resolution tracking layers (Muon Identifier). The design, construction, testing and expected run parameters of both the muon tracker and the muon identifier are described.

  8. Inheritance of the yeast mitochondrial genome

    DEFF Research Database (Denmark)

    Piskur, Jure

    1994-01-01

    Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast......Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast...

  9. The European Union and armed drones: framing the debate

    DEFF Research Database (Denmark)

    Martins, Bruno Oliveira

    2015-01-01

    Armed drones are an issue extremely relevant for the EU. The recent emergence of targeted killings as a common counter-terrorism technique, the existence of several EU member states using armed and surveillance drones in military scenarios, the presence of member states troops in areas where armed...... drones have been active, the US use of European-originated intelligence to execute targeted killings, and the broader status of international law, are developments that illustrate the importance of the topic. Yet, the EU still does not have an official position on armed drones. In 2014 the European...... Parliament recognized that this is problematic, adopting a Resolution that expressed “grave concern over the use of armed drones outside the international legal framework” and that urged the EU to “develop an appropriate policy response at both European and global level”. This Forum answers to the European...

  10. A Unified Approach for Reporting ARM Measurement Uncertainties Technical Report

    Energy Technology Data Exchange (ETDEWEB)

    Campos, E [Argonne National Laboratory; Sisterson, DL [Argonne National Laboratory

    2015-10-01

    The Atmospheric Radiation Measurement (ARM) Climate Research Facility is observationally based, and quantifying the uncertainty of its measurements is critically important. With over 300 widely differing instruments providing over 2,500 datastreams, concise expression of measurement uncertainty is quite challenging. The ARM Facility currently provides data and supporting metadata (information about the data or data quality) to its users through a number of sources. Because the continued success of the ARM Facility depends on the known quality of its measurements, the Facility relies on instrument mentors and the ARM Data Quality Office (DQO) to ensure, assess, and report measurement quality. Therefore, an easily-accessible, well-articulated estimate of ARM measurement uncertainty is needed.

  11. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  12. Yeasts preservation: alternatives for lyophilisation

    NARCIS (Netherlands)

    Nyanga, L.K.; Nout, M.J.R.; Smid, E.J.; Boekhout, T.; Zwietering, M.H.

    2012-01-01

    The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts duri

  13. A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker's Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast.

    Directory of Open Access Journals (Sweden)

    Chao Ran

    Full Text Available Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker's yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker's yeast were added to basal diets high in fishmeal and low in soybean (diet A or low in fishmeal and high in soybean (diet B, which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P < 0.05, and tended to improve FCR (P = 0.06 of fish compared to the control (no yeast. No significant differences were observed between inactivated yeast and control. Live yeast improved gut microvilli length (P < 0.001 and density (P < 0.05 while inactivated yeast did not. The hsp70 expression level in both the intestine and head kidney of fish was significantly reduced by live yeast (P < 0.05 but not inactivated yeast. Live yeast but not inactivated yeast reduced intestinal expression of tnfα (P < 0.05, tgfβ (P < 0.05 under diet A and il1β (P = 0.08. Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P < 0.001, indicating protection of the host against infection by A. hydrophila. In conclusion, secretory metabolites did not play major roles in the growth promotion and disease protection effects of yeast. Nevertheless, secretory metabolites were the major contributing factor towards improved gut

  14. Phage and Yeast Display.

    Science.gov (United States)

    Sheehan, Jared; Marasco, Wayne A

    2015-02-01

    Despite the availability of antimicrobial drugs, the continued development of microbial resistance--established through escape mutations and the emergence of resistant strains--limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies--and their encoding genes--against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases. PMID:26104550

  15. Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast.

    Science.gov (United States)

    Baro, Nicholas K; Pooput, Chaya; Roepe, Paul D

    2011-08-01

    Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.

  16. Eighteen new oleaginous yeast species.

    Science.gov (United States)

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes. PMID:27072563

  17. Modernization of African Armed Forces

    DEFF Research Database (Denmark)

    Mandrup, Thomas

    2015-01-01

    Concept paper framing the debate at the Dakar Forum Workshop on Modernization of Armed forces in Africa.......Concept paper framing the debate at the Dakar Forum Workshop on Modernization of Armed forces in Africa....

  18. Analysis of Arabidopsis glutathione-transferases in yeast.

    Science.gov (United States)

    Krajewski, Matthias P; Kanawati, Basem; Fekete, Agnes; Kowalski, Natalie; Schmitt-Kopplin, Philippe; Grill, Erwin

    2013-07-01

    The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and

  19. The economics of ribosome biosynthesis in yeast.

    Science.gov (United States)

    Warner, J R

    1999-11-01

    In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs). Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well. PMID:10542411

  20. The yeast Ski complex is a hetero-tetramer

    NARCIS (Netherlands)

    Synowsky, S.A.; Heck, A.J.R.

    2008-01-01

    The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we unambig

  1. Method for using a yeast alpha-amylase promoter

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Johnway (Richland, WA); Skeen, Rodney S. (Pendleton, OR); Hooker, Brian S. (Kennewick, WA); Anderson, Daniel B. (Pasco, WA)

    2003-04-22

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  2. BIOSYNTHESIS OF YEAST CAROTENOIDS

    Science.gov (United States)

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  3. Genetic study on yeast

    International Nuclear Information System (INIS)

    Research during the past year has moved ahead on several fronts. A major compilation of all the genetic mapping data for the yeast Saccharomyces cerevisiae has been completed. The map describes the location of over 300 genes on 17 chromosomes. A report on this work will appear in Microbiological Reviews in December 1980. Recombinant DNA procedures have been introduced into the experiments and RAD52 (one of the genes involved in recombination and repair damage), has been successfully cloned. This clone will be used to determine the gene product. Diploid cells homozygous for RAD52 have exceptionally high frequencies of mitotic loss of chromosomes. This loss is stimulated by ionizing radiation. This effect is a very significant finding. The effect has also been seen with certain other RAD mutants

  4. Lager yeast comes of age.

    Science.gov (United States)

    Wendland, Jürgen

    2014-10-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This "web of life" recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  5. Cadmium, ATPase-P, yeast. From transport to toxicity

    International Nuclear Information System (INIS)

    Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd2+-ATPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport by CadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using ATP hydrolysis as energy source. Directed mutagenesis of the membranous polar, sulphur and charged amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C354, C356 and proline P355 of the CPC motif located in Tm6, aspartate D692 in Tm8, glutamate E164 in Tm4 and methionine M149 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis ATP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to 100 μm cadmium whereas CadA expressing yeast cannot grow with 1 μm cadmium in the culture medium. This cadmium sensitivity was due to the localisation of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment produces an accumulation of mis-folded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs of toxicity. (author)

  6. 21 CFR 172.896 - Dried yeasts.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic...

  7. LIPASES PRODUCED BY YEASTS: POWERFUL BIOCATALYSTS FOR INDUSTRIAL PURPOSES

    Directory of Open Access Journals (Sweden)

    Luiza Lux Lock

    2007-12-01

    Full Text Available The term “lipolytic enzymes” refers to the lipases and carboxylic ester hydrolases. Lipase production is widespread among yeasts, but few are capable of producing lipases with interesting characteristics and in sufficient amounts to be industrially useful. The literature concerning lipases produced by Candida rugosa, Yarrowia (Candida lipolytica, Candida antarctica and other emerging lipase-producing yeasts is reviewed. The use of recombinant lipases is discussed, with emphasis on the utilization of heterologous expression systems and design of chimeras. Finally, the three approaches that aim the improvement of lipase production or the modification of the substrate selectivity of the enzyme (medium engineering, biocatalyst engineering, and protein engineering are discussed.

  8. Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Akihito; Bae, Jun Gu; Fukai, Kotaro; Tokumoto, Naoki; Kuroda, Kouichi; Ogawa, Jun; Shimizu, Sakayu; Ueda, Mitsuyoshi [Kyoto Univ. (Japan). Div. of Applied Life Sciences; Nakatani, Masato [Daiwa Kasei, Shiga (Japan)

    2012-05-15

    A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2{sup '}-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase. (orig.)

  9. ARM User Survey Report

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, LR

    2010-06-22

    The objective of this survey was to obtain user feedback to, among other things, determine how to organize the exponentially growing data within the Atmospheric Radiation Measurement (ARM) Climate Research Facility, and identify users’ preferred data analysis system. The survey findings appear to have met this objective, having received approximately 300 responses that give insight into the type of work users perform, usage of the data, percentage of data analysis users might perform on an ARM-hosted computing resource, downloading volume level where users begin having reservations, opinion about usage if given more powerful computing resources (including ability to manipulate data), types of tools that would be most beneficial to them, preferred programming language and data analysis system, level of importance for certain types of capabilities, and finally, level of interest in participating in a code-sharing community.

  10. Robot arm apparatus

    Science.gov (United States)

    Nachbar, Henry D.

    1992-01-01

    A robot arm apparatus is provided for inspecting and/or maintaining an interior of a steam generator which has an outside wall and a port for accessing the interior of the steam generator. The robot arm apparatus includes a flexible movable conduit for conveying inspection and/or maintenance apparatus from outside the steam generator to the interior of the steam generator. The flexible conduit has a terminal working end which is translated into and around the interior of the steam generator. Three motors located outside the steam generator are employed for moving the terminal working end inside the steam generator in "x", "y", and "z" directions, respectively. Commonly conducted inspection and maintenance operations include visual inspection for damaged areas, water jet lancing for cleaning sludge deposits, core boring for obtaining sludge deposits, and scrubbing of internal parts.

  11. Reconstruction of the yeast 2 micron plasmid partitioning mechanism.

    OpenAIRE

    Dobson, M J; Yull, F E; Molina, M.; Kingsman, S M; Kingsman, A J

    1988-01-01

    The effect of the yeast 2 micron circle encoded REP1 and REP2 gene products on plasmid partitioning and copy number control was analyzed by removing the open reading frames from their normal sequence context and transcriptional control regions and directing their expression using heterologous promoters in [cir0] host strains. Both the REP1 and REP2 gene products are directly required at appropriate levels of expression to reconstitute the 2 microns circle partitioning system in conjunction wi...

  12. A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker's Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast.

    Science.gov (United States)

    Ran, Chao; Huang, Lu; Liu, Zhi; Xu, Li; Yang, Yalin; Tacon, Philippe; Auclair, Eric; Zhou, Zhigang

    2015-01-01

    Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker's yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker's yeast were added to basal diets high in fishmeal and low in soybean (diet A) or low in fishmeal and high in soybean (diet B), which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP) activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P diet A) and il1β (P = 0.08). Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P diets supplemented with baker's yeast. PMID:26696403

  13. Kiikuv maja / Anu Arm

    Index Scriptorium Estoniae

    Arm, Anu

    2006-01-01

    Eesti Kunstiakadeemia esimese kursuse arhitektuuriüliõpilaste II semestri töö. Juhendaja arhitekt Andres Alver, ehitamise Pedaspeale organiseeris suvepraktika juhendaja arhitekt Jaan Tiidemann. Autor Anu Arm, kaasa töötasid ja valmis ehitasid: Ott Alver, Maarja Elm, Mari Hunt, Alvin Järving, Marten Kaevats, Riho Kerge, Reedik Poopuu, Anu Põime, Helen Rebane, Kaisa Saarva, Martin Tago, Reet Volt. Valmis: 19. VIII 2006

  14. Marine Yeasts and Their Applications in Mariculture

    Institute of Scientific and Technical Information of China (English)

    CHI Zhenming; LIU Zhiqiang; GAO Lingmei; GONG Fang; MA Chunling; WANG Xianghong; LI Haifeng

    2006-01-01

    The terrestrial yeasts have been receiving great attention in science and industry for over one hundred years because they can produce many kinds of bioactive substances. However, little is known about the bioactive substances of marine yeasts. In recent years, it has been found that marine yeasts have wide applications in mariculture and other fields.Therefore, marine yeasts, the bioactive substances from them and the applications of marine yeasts themselves and the bioactive substances they produced are reviewed in this paper.

  15. Genetically engineered yeast

    DEFF Research Database (Denmark)

    2014-01-01

    A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate semialde......A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate...

  16. Vaginal Yeast Infections (For Parents)

    Science.gov (United States)

    ... infection caused by a type of fungus called candida albicans . Yeast infections usually happen in warm, moist parts of the ... fungus can grow. Doctors call this candida overgrowth candidiasis (pronounced: can-dih-DYE-uh-sis) Candida can ...

  17. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  18. Reconstitution of the mitochondrial calcium uniporter in yeast.

    Science.gov (United States)

    Kovács-Bogdán, Erika; Sancak, Yasemin; Kamer, Kimberli J; Plovanich, Molly; Jambhekar, Ashwini; Huber, Robert J; Myre, Michael A; Blower, Michael D; Mootha, Vamsi K

    2014-06-17

    The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery. PMID:24889638

  19. Reconstitution of the mitochondrial calcium uniporter in yeast.

    Science.gov (United States)

    Kovács-Bogdán, Erika; Sancak, Yasemin; Kamer, Kimberli J; Plovanich, Molly; Jambhekar, Ashwini; Huber, Robert J; Myre, Michael A; Blower, Michael D; Mootha, Vamsi K

    2014-06-17

    The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

  20. Sociobiology of the budding yeast

    Indian Academy of Sciences (India)

    Dominika M Wloch-Salamon

    2014-04-01

    Social theory has provided a useful framework for research with microorganisms. Here I describe the advantages and possible risks of using a well-known model organism, the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitness-based Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence of apoptosis.

  1. Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and {beta}-glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Apiwatanapiwat, Waraporn; Rugthaworn, Prapassorn [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Kasetsart Univ., Bangkok (Thailand). Nanotechnology and Biotechnology Div.; Murata, Yoshinori; Kosugi, Akihiko; Arai, Takamitsu; Mori, Yutaka [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Yamada, Ryosuke; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering

    2011-04-15

    In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying {alpha}-amylase ({alpha}-AM), glucoamylase, endoglucanase, cellobiohydrase, and {beta}-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley {beta}-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes. (orig.)

  2. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    Directory of Open Access Journals (Sweden)

    Fernando eAleman

    2014-09-01

    Full Text Available Potassium (K+ is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter.

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

    Science.gov (United States)

    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.

  4. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    Directory of Open Access Journals (Sweden)

    Chang Jui-Jen

    2012-07-01

    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  5. Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus(HCV)by cloning the gene of NS5ABP37 protein into pGBKT7,then the recombinant plasmid DNA was transformed into yeast AH109(α type).The transformed yeast AH109 was mated with yeast Y187(α type)containing liver cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout ...

  6. Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers

    DEFF Research Database (Denmark)

    Haaning, J; Oxvig, C; Overgaard, Michael Toft;

    1997-01-01

    by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature......Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris...

  7. Indole-3-acetic acid-producing yeasts in the phyllosphere of the carnivorous plant Drosera indica L.

    Directory of Open Access Journals (Sweden)

    Pei-Feng Sun

    Full Text Available Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.

  8. Indole-3-acetic acid-producing yeasts in the phyllosphere of the carnivorous plant Drosera indica L.

    Science.gov (United States)

    Sun, Pei-Feng; Fang, Wei-Ta; Shin, Li-Ying; Wei, Jyuan-Yu; Fu, Shih-Feng; Chou, Jui-Yu

    2014-01-01

    Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture. PMID:25464336

  9. Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.

    Directory of Open Access Journals (Sweden)

    Mariana Aprigio Assis-Marques

    Full Text Available The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM. It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

  10. Kidins220/ARMS Is a Novel Modulator of Short-Term Synaptic Plasticity in Hippocampal GABAergic Neurons

    OpenAIRE

    Joachim Scholz-Starke; Fabrizia Cesca; Giampietro Schiavo; Fabio Benfenati; Pietro Baldelli

    2012-01-01

    Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a scaffold protein highly expressed in the nervous system. Previous work on neurons with altered Kidins220/ARMS expression suggested that this protein plays multiple roles in synaptic function. In this study, we analyzed the effects of Kidins220/ARMS ablation on basal synaptic transmission and on a variety of short-term plasticity paradigms in both excitatory and inhibitory synapses using a r...

  11. LISA Long-Arm Interferometry

    Science.gov (United States)

    Thorpe, James I.

    2009-01-01

    An overview of LISA Long-Arm Interferometry is presented. The contents include: 1) LISA Interferometry; 2) Constellation Design; 3) Telescope Design; 4) Constellation Acquisition; 5) Mechanisms; 6) Optical Bench Design; 7) Phase Measurement Subsystem; 8) Phasemeter Demonstration; 9) Time Delay Interferometry; 10) TDI Limitations; 11) Active Frequency Stabilization; 12) Spacecraft Level Stabilization; 13) Arm-Locking; and 14) Embarassment of Riches.

  12. Emulsifying activity of hydrocarbonoclastic marine yeasts

    Digital Repository Service at National Institute of Oceanography (India)

    Gupta, R.

    Marine yeast growth on four petroleum hydrocarbons induced the production of extracellular emulsifying agents (biosurfactants). Out of the 17 marine yeast isolates tested, 7 isolates, i.e., Candida parapsilosis, C. cantarelli, C. membranae...

  13. INVESTIGATIONS INTO MAGNESIUM BIOSORPTION BY WASTE BREWERY YEAST SACCHAROMYCES UVARUM

    Directory of Open Access Journals (Sweden)

    Małgorzata Gniewosz

    2007-03-01

    Full Text Available Investigations were carried out into the capacity of waste brewery yeast Saccharomyces uvarum for biosorption of magnesium originated from a solution of dehydrated salt of magnesium chloride, depending on the number of cells and diferent pH of the suspension during 6 hours. The concentration of MgCl2•6H2O in the solution was adjusted so as to maintain a stable content of magnesium as expressed per pure element, i.e. 1.25 g/dm3 of solution. In the first stage, the number of cells was differentiated in yeast slurry through either condensation or dilution. In the second stage, pH of yeast suspension was differentiated (pH 5.5, 6.0 and 7.0 at a constant number of cells. The solutions examined were kept under anaerobic and aerobic conditions. Determination of magnesium content of yeast biomass was carried out with the method of atomic adsorption spectroscopy after 15 min, 1 h, 2 h, 4 h and 6 h of experiments. The highest content of magnesium (13.76 mg/g d.m. was obtained at the lowest number of cells in the solution, i.e. 3.5 × 108 cells/cm3 under aerobic conditions. An increase in solution pH facilitated biosorption of magnesium by the yeast. At pH 7.0, after 6 hours of the experiment, the yeasts contained 15.19 mg Mg/g d.m. when kept under anaerobic conditions and 17.22 mg Mg/g d.m. when kept under aerobic conditions.

  14. Characterisation of the DNA binding domain of the yeast RAP1 protein

    OpenAIRE

    Henry, Y A; Chambers, A.; Tsang, J S; Kingsman, A J; Kingsman, S M

    1990-01-01

    The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'heli...

  15. Robotic Arm Biobarrier Cable

    Science.gov (United States)

    2008-01-01

    This image, taken by the Surface Stereo Imager on NASA's Phoenix Mars Lander on the 14th Martian day of the mission (June 7, 2008), shows the cable that held the Robotic Arm's biobarrier in place during flight has snapped. The cable's springs retracted to release the biobarrier right after landing. To the lower right of the image a spring is visible. Extending from that spring is a length of cable that snapped during the biobarrier's release. A second spring separated from the cable when it snapped and has been photographed on the ground under the lander near one of the legs. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  16. Construction of cDNA subtractive library between yeast and mycelium phase of Sporothrix schenckii and screening of differently expressed genes about dimorphic transition%申克孢子丝菌菌丝相和酵母相cDNA消减文库的构建及差异表达基因的筛选

    Institute of Scientific and Technical Information of China (English)

    周汛; 杨致邦; 肖异珠

    2011-01-01

    The purpose of this study was to screen the differentially expressed genes about dimorphic transition of Sporothrix schenckii by construction of eDNA subtractive library between yeast and mycelium phase.The eDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.Of 751 and 875 ESTs were obtained in M+Y and Y+M library, separately.After splicing of ESTs, 101 and 249 unigenes were obtained in M +Y and Y+ M libraries.During the construction of cDNA subtractive libraries, the distribution of differently expressed genes varied with dimorphic transition.The over expressed genes with diversity and complexity were divided into structural genes,metabolic enzymes, molecule on cell surface and molecule with indistinct function.Results disclose that construction of cDNA subtractive libraries for the dimorphic transition and bioinformatics analysis for those differently expressed genes lay the foundation for screening the related genes involved in the pathogenesis of Sporothrix schenckii infection.%目的 构建申克孢子丝菌双相cDNA消减文库,筛选与双相转换相关的差异表达基因.方法 运用抑制性消减杂交技术,构建申克孢子丝菌菌丝相(Mycelium,M)和酵母相(Yeast,Y)的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M+Y文库获得751条表达序列标签(Expressed Sequence Tags,ESTs),经拼接后获得101条unigenes;Y+M文库获得875条ESTs,拼接获得249条unigenes.申克孢子丝菌酵母相菌丝相的转换伴随着不同菌相细胞差异基因的高表达,这些高表达的差异基因可分为结构基因类、代谢酶类、细胞表面分子类及功能不明的细胞分子.结论 成功构建了申克孢子丝菌双相转换相关的cDNA消减文库基础上,筛选出部分差异表达基

  17. 21 CFR 73.355 - Phaffia yeast.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Phaffia yeast. 73.355 Section 73.355 Food and... ADDITIVES EXEMPT FROM CERTIFICATION Foods § 73.355 Phaffia yeast. (a) Identity. (1) The color additive phaffia yeast consists of the killed, dried cells of a nonpathogenic and nontoxicogenic strain of...

  18. Transcriptional robustness and protein interactions are associated in yeast

    Directory of Open Access Journals (Sweden)

    Conant Gavin C

    2011-05-01

    Full Text Available Abstract Background Robustness to insults, both external and internal, is a characteristic feature of life. One level of biological organization for which noise and robustness have been extensively studied is gene expression. Cells have a variety of mechanisms for buffering noise in gene expression, but it is not completely clear what rules govern whether or not a given gene uses such tools to maintain appropriate expression. Results Here, we show a general association between the degree to which yeast cells have evolved mechanisms to buffer changes in gene expression and whether they possess protein-protein interactions. We argue that this effect bears an affinity to epistasis, because yeast appears to have evolved regulatory mechanisms such that distant changes in gene copy number for a protein-protein interaction partner gene can alter a gene's expression. This association is not unexpected given recent work linking epistasis and the deleterious effects of changes in gene dosage (i.e., the dosage balance hypothesis. Using gene expression data from artificial aneuploid strains of bakers' yeast, we found that genes coding for proteins that physically interact with other proteins show less expression variation in response to aneuploidy than do other genes. This effect is even more pronounced for genes whose products interact with proteins encoded on aneuploid chromosomes. We further found that genes targeted by transcription factors encoded on aneuploid chromosomes were more likely to change in expression after aneuploidy. Conclusions We suggest that these observations can be best understood as resulting from the higher fitness cost of misexpression in epistatic genes and a commensurate greater regulatory control of them.

  19. Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast.

    OpenAIRE

    Karin, M; Najarian, R; Haslinger, A; Valenzuela, P; Welch, J; Fogel, S

    1984-01-01

    Copper resistance in yeast is controlled by the CUP1 locus. The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies. To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence. We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains. One of those transcription ...

  20. Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.

    OpenAIRE

    Ketner, G; Spencer, F; Tugendreich, S; C. Connelly; Hieter, P

    1994-01-01

    A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the Y...

  1. Functional Distinctions between IMP Dehydrogenase Genes in Providing Mycophenolate Resistance and Guanine Prototrophy to Yeast*

    OpenAIRE

    Hyle, Judith W.; Shaw, Randal J.; Reines, Daniel

    2003-01-01

    IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show...

  2. CoopTFD: a repository for predicted yeast cooperative transcription factor pairs

    OpenAIRE

    Wu, Wei-Sheng; Lai, Fu-Jou; Tu, Bor-Wen; Chang, Darby Tien-Hao

    2016-01-01

    In eukaryotic cells, transcriptional regulation of gene expression is usually accomplished by cooperative Transcription Factors (TFs). Therefore, knowing cooperative TFs is helpful for uncovering the mechanisms of transcriptional regulation. In yeast, many cooperative TF pairs have been predicted by various algorithms in the literature. However, until now, there is still no database which collects the predicted yeast cooperative TFs from existing algorithms. This prompts us to construct Coope...

  3. Comparative Evaluation of the BD Phoenix Yeast ID Panel and Remel RapID Yeast Plus System for Yeast Identification

    OpenAIRE

    Michelle L. Grant; Shobha Parajuli; Raquel Deleon-Gonsalves; Raghava Potula; Truant, Allan L.

    2016-01-01

    Becton Dickinson Phoenix Yeast ID Panel was compared to the Remel RapID Yeast Plus System using 150 recent clinical yeast isolates and the API 20C AUX system to resolve discrepant results. The concordance rate between the Yeast ID Panel and the RapID Yeast Plus System (without arbitration) was 93.3% with 97.3% (146/150) and 95.3% (143/150) of the isolates correctly identified by the Becton Dickinson Phoenix and the Remel RapID, respectively, with arbitration.

  4. Calling Card Analysis in Budding Yeast.

    Science.gov (United States)

    Mayhew, David; Mitra, Robi D

    2016-02-01

    Calling card analysis is a high-throughput method for identifying the genomic binding sites of multiple transcription factors in a single experiment in budding yeast. By tagging a DNA-binding protein with a targeting domain that directs the insertion of the Ty5 retrotransposon, the genomic binding sites for that transcription factor are marked. The transposition locations are then identified en masse by Illumina sequencing. The calling card protocol allows for simultaneous analysis of multiple transcription factors. By cloning barcodes into the Ty5 transposon, it is possible to pair a unique barcode with every transcription factor in the experiment. The method presented here uses expression of transcription factors from their native loci; however, it can also be altered to measure binding sites of transcription factors overexpressed from a plasmid. PMID:26832687

  5. 中国虎纹捕鸟蛛神经毒素肽基因的克隆及在酵母中的表达%Clonging and Expression of a Neurotoxin Peptide Gene from Chinese Bird Spider (Selenocosmia huwena) in Yeast

    Institute of Scientific and Technical Information of China (English)

    李敏; 聂东宋

    2006-01-01

    Huwentoxin- Ⅰ ( HWTX- Ⅰ ) is a 33 amino acids peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. HWTX- Ⅰ acts as a presynaptic toxin that blocks the high-voltage activated calcium channel. HWTX- Ⅰ can also affect the release of acetylcholine and adrenalin of sympathetic-vagus nerve terminal by electrical stimulation. The result indicates that HWTX- Ⅰ is a potential analgesic drug candidate. In this report, the sequence of cDNA of HWTX- Ⅰ was identified by RT-PCR and further successfully expressed HWTX-1 peptide in Pichia pastoris expression vector pPIC9K using his4 mutant yeast GS115. The His+ Mut8 clones were screened and recombinant HWTX- Ⅰ was secreted into the culture media using methanol as an inducer. The expressed HWTX-1 product was further analyzed and its biological activities were confirmed by some biological assays. Similar physiological activity was observed in the yeast-expressed HWTX-1 compared with the native HWTX- Ⅰ . Fig 4, Ref 10%从中国珍稀毒蜘蛛种虎纹捕鸟蛛的粗毒中分离纯化的虎纹捕鸟蛛毒素-Ⅰ(HWTX-Ⅰ)是含33个氨基酸的多肽.有关实验已证实,HWTX-Ⅰ是一种作用于突触前膜的神经毒素和高阈值钙通道抑制剂,在电刺激时HWTX-Ⅰ也影响交感神经释放肾上腺素和迷走神经释放乙酰胆碱,这些结果表明,HWTX-Ⅰ具有潜在的镇痛活性.本文报道通过RT-PCR方法克隆、测定了HWTX-Ⅰ的cDNA序列.在此基础上,以pPIC9K为表达载体和GS115为宿主,筛选His+Muts克隆,以甲醇为诱导剂,成功地构建并在毕赤酵母系统分泌表达HWTX-Ⅰ基因.进一步分析HWTX-Ⅰ表达产物,生物活性实验观察到酵母表达体系所获得的HWTX-Ⅰ产物具有与天然HWTX-Ⅰ相似的生理活性.图4参10

  6. Yeast as factory and factotum.

    Science.gov (United States)

    Dixon, B

    2000-02-01

    After centuries of vigorous activity in making fine wines, beers and breads, Saccharomyces cerevisiae is now acquiring a rich new portfolio of skills, bestowed by genetic manipulation. As shown in a recent shop-window of research supported by the European Commission, yeasts will soon be benefiting industries as diverse as fish farming, pharmaceuticals and laundering.

  7. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  8. Black yeasts in cold habitats

    NARCIS (Netherlands)

    L. Selbmann; G.S. de Hoog; L. Zucconi; D. Isola; S. Onofri

    2014-01-01

    Black yeasts have already been known since the end of the nineteenth century, but for a number of reasons, only few workers were familiar with them. That was since recently, until the wealth of biodiversity, stunning ecologies and potential applications have become apparent. Some remote and extreme

  9. Fermentation of lactose by yeast cells secreting recombinant fungal lactase.

    OpenAIRE

    Ramakrishnan, S.; Hartley, B S

    1993-01-01

    Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic g...

  10. Yeast as a Heterologous Model System to Uncover Type III Effector Function

    Science.gov (United States)

    Popa, Crina; Coll, Núria S.; Valls, Marc; Sessa, Guido

    2016-01-01

    Type III effectors (T3E) are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. “Favourite” targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK) signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure–function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations of S

  11. Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

    Science.gov (United States)

    Tehseen, Muhammad; Dumancic, Mira; Briggs, Lyndall; Wang, Jian; Berna, Amalia; Anderson, Alisha; Trowell, Stephen

    2014-01-01

    Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) triple mutant ("Cyb") with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.

  12. Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

    Directory of Open Access Journals (Sweden)

    Muhammad Tehseen

    Full Text Available Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1 of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ triple mutant ("Cyb" with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.

  13. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  14. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Directory of Open Access Journals (Sweden)

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  15. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.

    Science.gov (United States)

    Schott, Daniel H; Collins, Ruth N; Bretscher, Anthony

    2002-01-01

    Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

  16. 'Yeast mail': a novel Saccharomyces application (NSA) to encrypt messages.

    Science.gov (United States)

    Rosemeyer, Helmut; Paululat, Achim; Heinisch, Jürgen J

    2014-09-01

    The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)-based, one-step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages.

  17. Advances in mechanisms and modifications for rendering yeast thermotolerance.

    Science.gov (United States)

    Gao, Liman; Liu, Yueqin; Sun, Hun; Li, Chun; Zhao, Zhiping; Liu, Guiyan

    2016-06-01

    Thermotolerant Saccharomyces cerevisiae is widely regarded as an attractive strain with which to accomplish the coupling of enzyme saccharification, ethanol fermentation and ethanol distillation in non-grain fuel bioethanol fermentation systems, and it has many advantages for increasing the ethanol yield and reducing production costs. This review provided an overview of the yeast heat-resistant mechanisms from six aspects, including gene expression responses, heat shock proteins, trehalose, ATPase, the ubiquitin-proteasome pathway and heat-induced antioxidant defenses. Innovative methods, such as random and rational strategies for improving yeast thermotolerance, were further discussed, and several special cases were provided. To rationally engineer thermotolerance in yeast, the advances in employing heat-resistant mechanisms of thermophiles were particularly discussed. By designing and constructing heat-resistant devices consists of heat-resistant parts from thermophiles to yeast, a superior thermotolerance of S. cerevisiae has been achieved, providing a new system with important applications for research regarding the improvement of the robustness of microbes. PMID:26685013

  18. Effect of chromosome tethering on nuclear organization in yeast.

    Directory of Open Access Journals (Sweden)

    Barış Avşaroğlu

    Full Text Available Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML on chromosome III in wild-type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.

  19. Construction of a lactose-assimilating strain of baker's yeast.

    Science.gov (United States)

    Adam, A C; Prieto, J A; Rubio-Texeira, M; Polaina, J

    1999-09-30

    A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey.

  20. ARM Lead Mentor Selection Process

    Energy Technology Data Exchange (ETDEWEB)

    Sisterson, DL

    2013-03-13

    The ARM Climate Research Facility currently operates more than 300 instrument systems that provide ground-based observations of the atmospheric column. To keep ARM at the forefront of climate observations, the ARM infrastructure depends heavily on instrument scientists and engineers, also known as Instrument Mentors. Instrument Mentors must have an excellent understanding of in situ and remote-sensing instrumentation theory and operation and have comprehensive knowledge of critical scale-dependent atmospheric processes. They also possess the technical and analytical skills to develop new data retrievals that provide innovative approaches for creating research-quality data sets.

  1. Opportunistic yeast pathogens: reservoirs, virulence mechanisms, and therapeutic strategies.

    Science.gov (United States)

    Polvi, Elizabeth J; Li, Xinliu; O'Meara, Teresa R; Leach, Michelle D; Cowen, Leah E

    2015-06-01

    Life-threatening invasive fungal infections are becoming increasingly common, at least in part due to the prevalence of medical interventions resulting in immunosuppression. Opportunistic fungal pathogens of humans exploit hosts that are immunocompromised, whether by immunosuppression or genetic predisposition, with infections originating from either commensal or environmental sources. Fungal pathogens are armed with an arsenal of traits that promote pathogenesis, including the ability to survive host physiological conditions and to switch between different morphological states. Despite the profound impact of fungal pathogens on human health worldwide, diagnostic strategies remain crude and treatment options are limited, with resistance to antifungal drugs on the rise. This review will focus on the global burden of fungal infections, the reservoirs of these pathogens, the traits of opportunistic yeast that lead to pathogenesis, host genetic susceptibilities, and the challenges that must be overcome to combat antifungal drug resistance and improve clinical outcome. PMID:25700837

  2. Arménie

    Directory of Open Access Journals (Sweden)

    François Verdier

    2012-04-01

    Full Text Available L’Arménie est une petite république du Caucase, à la limite sud–est de l’Europe, qui a gagné son autonomie en 1990 après l’ouverture du bloc soviétique. Le nouveau Ministère du Patrimoine a sollicité la coopération de la France pour mettre en place une nouvelle politique culturelle. Tout d’abord, une évaluation sur place de la situation dans les domaines des monuments historiques, de l’archéologie et de l’Inventaire a permis d’envisager les réponses à proposer. Pour la demande d’informatisation des dossiers d’inventaire déjà réalisés sous l’autorité de l’Académie de Saint–Petersbourg, nous avons proposé de former des chercheurs arméniens aux méthodes et techniques de l’Inventaire général. L’accueil d’une stagiaire pendant trois mois au service régional de l’Inventaire de Haute–Normandie a été suivi par la mise en place d’un équipement informatique à Yérévan, puis par l’accueil et la formation de techniciens informaticiens et photographes arméniens. De retour dans leur pays ils ont commencé à remettre en place un service d’inventaire dont le programme comprend la création d’une base de données patrimoniales, le recensement de la ville de Yérévan, la numérisation d’images pour la publication d’un indicateur du patrimoine et la préparation de dossiers de protection au titre du patrimoine mondial.The Armenian heritage comprises both archaeological remains of towns destroyed by never–ending wars and a number of old churches from the eleventh and twelfth centuries. Yerevan, the capital of Armenia, was founded three thousand years ago and is one of Europe’s oldest capitals. From 1925 it has developed according to an ambitious urban planning project. After the major political upheavals of 1991, a special ministry was created to look after the architectural and movable heritage of the country and to promote the Armenian national identity. A mission in Yerevan was

  3. Merozoite surface protein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of cholera toxin B subunit glycoprotein expressed in yeast induced protective immunity against lethal malaria infection in mice.

    Science.gov (United States)

    Miyata, Takeshi; Harakuni, Tetsuya; Taira, Toki; Matsuzaki, Goro; Arakawa, Takeshi

    2012-01-20

    Methylotrophic yeast (Pichia pastoris) secreted cholera toxin B subunit (CTB) predominantly as a biologically active pentamer (PpCTB) with identical ganglioside binding affinity profiles to that of choleragenoid. Unlike choleragenoid, however, the PpCTB did not induce a footpad edema response in mice. Of the two potential glycosylation sites (NIT(4-6) and NKT(90-92)) for this protein, a N-linked oligosaccharide was identified at Asn4. The oligosaccharide, presumed to extend from the lateral circumference of the CTB pentamer ring structure, was exploited as a site-specific anchoring scaffold for the C-terminal 19-kDa merozoite surface protein-1 (MSP1-19) of the rodent malaria parasite, Plasmodium yoelii. Conjugation of MSP1-19 to PpCTB via its oligosaccharide moiety induced higher protective efficacy against lethal parasite infection than conjugation directly to the PpCTB protein body in both intranasal and subcutaneous immunization regimes. Such increased protection was potentially due to the higher antigen loading capacity of CTB achieved when the antigen was linked to the extended branches of the oligosaccharide. This might have allowed the antigen to reside in more spacious molecular environment with less steric hindrance between the constituent molecules of the fusion complex. PMID:22119928

  4. The wine and beer yeast Dekkera bruxellensis.

    Science.gov (United States)

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history.

  5. An IPTG-inducible derivative of the fission yeast nmt promoter

    DEFF Research Database (Denmark)

    Kjærulff, Søren; Nielsen, Olaf

    2015-01-01

    We here describe an IPTG-inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly...

  6. Metabolic regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast

    NARCIS (Netherlands)

    Bouwman, Jildau; Kiewiet, Jose; Lindenbergh, Alexander; van Eunen, Karen; Siderius, Marco; Bakker, Barbara M.

    2011-01-01

    Intracellular accumulation of glycerol is essential for yeast cells to survive hyperosmotic stress. Upon hyperosmotic stress the gene expression of enzymes in the glycerol pathway is strongly induced. Recently, however, it was shown that this gene-expression response is not essential for survival of

  7. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    Science.gov (United States)

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  8. 申克孢子丝菌酵母相与白念珠菌诱导中性粒细胞活性氧簇的差异性表达%Differential expression of reactive oxygen species in human neutrophils stimulated by the yeast phase of Sporothrix Schenckii and Candida albicans

    Institute of Scientific and Technical Information of China (English)

    董碧麟; 李东升; 柳卫凰; 童中胜; 陈柳青; 段逸群

    2013-01-01

    Objective To compare the reactive oxygen species (ROS) expression in human neutrophils phagocytizing Sporothrix Schenckii and Candida albicans yeast cells,and to compare the fungicidal activity of human neutrophils against Sporothrix Schenckii and Candida albicans.Methods Human neutrophils were isolated from peripheral blood by using density gradient centrifugation method,and cultured with the presence of the yeast phase of a Sporothrix Schenckii clinical isolate and a standard strain of Candida albicans (ATCC 90028)at a multiplicity of infection of 10 or 1 for 60-210 minutes.Subsequently,flow cytometry with ROS probe (2',7'-dichlorofluorescein diacetate,DCFH-DA) was carried out to for the real time detection of intracellular ROS level,confocal laser scanning microscopy (CLSM) for the observation of ROS distribution.In addition,the fungicidal efficiency of neutrophils against Sporothrix Schenckii and Candida albicans was estimated by the number of colonies after additional culture of neutrophil lysates on brain-heart infusion agar (BHIA) medium.Statistical analysis was done by using univariate analysis of variance and LSD-t test.Results The intracellular ROS level peaked at 60 minutes in neutrophils incubated with Sporothrix Schenckii yeast cells,then decreased rapidly from 60 minutes to 210 minutes.Compared with the neutrophils incubated with Candida albicans yeast cells,those with Sporothrix Schenckii yeast cells showed a higher ROS level (expressed as mean fluorescence intensity) at 60minutes (159.67 ± 11.34 vs.112.22 ± 9.66,P< 0.01),but a lower ROS level at 120 minutes (89.01 ± 9.81 vs.110.25 ± 7.28,P< 0.05) and 180 minutes (57.63 ± 8.46 vs.109.98 ± 9.00,P< 0.01).CLSM revealed that ROS was mainly distributed in neutrophils with phagocytized fungal spores,and especially on the surface of phagocytized spores.Furthermore,the percentage of yeast cells killed by neutrophils was significantly lower for Sporothrix Schenckii than for Candida albicans at 180

  9. Arms control: misplaced focus

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, W.A.; Derber, C.

    1986-03-01

    Most of the nuclear debate consists of arguments about which weapons systems should be built, controlled, canceled, frozen, or retired. Short of virtually complete, multilateral nuclear disarmament, however, no change in the pace, balance, or even the direction of the arms race can make much difference in the risk of nuclear war, the damage should one occur, or the division of international political power. This includes Star Wars, the nuclear freeze, and even large cuts in or stabilization of offensive nuclear arsenals. A better starting point for nuclear politics would be the insight that nuclear weapons have completely changed the logic of power as it has been handed down through the ages. Military force, perfected to its highest level, has invalidated itself - for in a nuclearized world, any resort to force by a nuclear power risks escalation to its ultimate level, and thus to oblivion for all. Trying to rationalize and control the ultimate force is far less realistic and important than limiting the provocation of conflict and the use of force at lower, non-nuclear levels - by the United States, it clients, and, to the extent possible, its adversaries. 13 references.

  10. ARM Standards Policy Committee Report

    Energy Technology Data Exchange (ETDEWEB)

    Cialella, A; Jensen, M; Koontz, A; McFarlane, S; McCoy, R; Monroe, J; Palanisamy, G; Perez, R; Sivaraman, C

    2012-09-19

    Data and metadata standards promote the consistent recording of information and are necessary to ensure the stability and high quality of Atmospheric Radiation Measurement (ARM) Climate Research Facility data products for scientific users. Standards also enable automated routines to be developed to examine data, which leads to more efficient operations and assessment of data quality. Although ARM Infrastructure agrees on the utility of data and metadata standards, there is significant confusion over the existing standards and the process for allowing the release of new data products with exceptions to the standards. The ARM Standards Policy Committee was initiated in March 2012 to develop a set of policies and best practices for ARM data and metadata standards.

  11. [Apothecaries' arms of Voltaire's family].

    Science.gov (United States)

    Chaigneau, M

    1998-01-01

    Apothecaries of Voltaire's family can be divided into two groups. Marceton Family: Claude Marceton, Hierosme and Pierre Testefolle, from Thouars. Arouet family: Jehan Arouet, Pierre Rochard and Jean Gougeard. Five of those apothecaries beand arms. PMID:11625327

  12. Antioxidant defense parameters as predictive biomarkers for fermentative capacity of active dried wine yeast.

    Science.gov (United States)

    Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

    2014-08-01

    The production of active dried yeast (ADY) is a common practice in industry for the maintenance of yeast starters and as a means of long term storage. The process, however, causes multiple cell injuries, with oxidative damage being one of the most important stresses. Consequentially, dehydration tolerance is a highly appreciated property in yeast for ADY production. In this study we analyzed the cellular redox environment in three Saccharomyces cerevisiae wine strains, which show markedly different fermentative capacities after dehydration. To measure/quantify the effect of dehydration on the S. cerevisiae strains, we used: (i) fluorescent probes; (ii) antioxidant enzyme activities; (ii) intracellular damage; (iii) antioxidant metabolites; and (iv) gene expression, to select a minimal set of biochemical parameters capable of predicting desiccation tolerance in wine yeasts. Our results show that naturally enhanced antioxidant defenses prevent oxidative damage after wine yeast biomass dehydration and improve fermentative capacity. Based on these results we chose four easily assayable parameters/biomarkers for the selection of industrial yeast strains of interest for ADY production: trehalose and glutathione levels, and glutathione reductase and catalase enzymatic activities. Yeast strains selected in accordance with this process display high levels of trehalose, low levels of oxidized glutathione, a high induction of glutathione reductase activity, as well as a high basal level and sufficient induction of catalase activity, which are properties inherent in superior ADY strains.

  13. Human yeast-specific CD8 T lymphocytes show a nonclassical effector molecule profile.

    Science.gov (United States)

    Breinig, Tanja; Scheller, Nicoletta; Glombitza, Birgit; Breinig, Frank; Meyerhans, Andreas

    2012-05-01

    Pathogenic yeast and fungi represent a major group of human pathogens. The consequences of infections are diverse and range from local, clinically uncomplicated mycosis of the skin to systemic, life-threatening sepsis. Despite extensive MHC class I-restricted frequencies of yeast-specific CD8 T lymphocytes in healthy individuals and the essential role of the cell-mediated immunity in controlling infections, the characteristics and defense mechanisms of antifungal effector cells are still unclear. Here, we describe the direct analysis of yeast-specific CD8 T lymphocytes in whole blood from healthy individuals. They show a unique, nonclassical phenotype expressing granulysin and granzyme K in lytic granules instead of the major effector molecules perforin and granzyme B. After stimulation in whole blood, yeast-specific CD8 T cells degranulated and, upon cultivation in the presence of IL-2, their granula were refilled with granulysin rather than with perforin and granzyme B. Moreover, yeast-specific stimulation through dendritic cells but not by yeast cells alone led to degranulation of the effector cells. As granulysin is the only effector molecule in lytic granules known to have antifungal properties, our data suggest yeast-specific CD8 T cells to be a nonclassical effector population whose antimicrobial effector machinery seems to be tailor-made for the efficient elimination of fungi as pathogens.

  14. Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough.

    Science.gov (United States)

    Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

    2012-05-01

    Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking. PMID:22280966

  15. Antioxidant defense parameters as predictive biomarkers for fermentative capacity of active dried wine yeast.

    Science.gov (United States)

    Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

    2014-08-01

    The production of active dried yeast (ADY) is a common practice in industry for the maintenance of yeast starters and as a means of long term storage. The process, however, causes multiple cell injuries, with oxidative damage being one of the most important stresses. Consequentially, dehydration tolerance is a highly appreciated property in yeast for ADY production. In this study we analyzed the cellular redox environment in three Saccharomyces cerevisiae wine strains, which show markedly different fermentative capacities after dehydration. To measure/quantify the effect of dehydration on the S. cerevisiae strains, we used: (i) fluorescent probes; (ii) antioxidant enzyme activities; (ii) intracellular damage; (iii) antioxidant metabolites; and (iv) gene expression, to select a minimal set of biochemical parameters capable of predicting desiccation tolerance in wine yeasts. Our results show that naturally enhanced antioxidant defenses prevent oxidative damage after wine yeast biomass dehydration and improve fermentative capacity. Based on these results we chose four easily assayable parameters/biomarkers for the selection of industrial yeast strains of interest for ADY production: trehalose and glutathione levels, and glutathione reductase and catalase enzymatic activities. Yeast strains selected in accordance with this process display high levels of trehalose, low levels of oxidized glutathione, a high induction of glutathione reductase activity, as well as a high basal level and sufficient induction of catalase activity, which are properties inherent in superior ADY strains. PMID:24644263

  16. A Three-Dimensional Model of the Yeast Genome

    Science.gov (United States)

    Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

    Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

  17. Bistable Head Positioning Arm Latch

    Science.gov (United States)

    Wasson, Ken; Endo, Juro; Mita, Masahiro; Abelein, Nathan

    A simple, low cost, yet effective device has been developed for immobilizing the head-arm assembly in a disk drive or similar mechanism during power-off conditions. The latching scheme also provides a consistent means of releasing the head-arm assembly from the immobilized position upon power up of the disk drive. The latch uses no electrical power in either immobilized or released state. This design is immune to extreme torque and linear shock forces applied to the disk drive case. The latch system can use the energy stored in the spinning disks to drive the head-arm assembly toward a safe position while simultaneously arming the latch mechanism to secure the head-arm assembly in the safe position upon arrival. A low energy five msec pulse of current drives the latch from one state to the other. Solenoids as presently used in latch mechanisms are bulky, expensive, have variable force characteristics, and often generate contaminants. The latch described in this paper is expected to replace such solenoids. It may also replace small magnet latches, which have limited latch force and apply unwanted torque to a proximate head positioning arm.

  18. Mycotoxins - prevention and decontamination by yeasts.

    Science.gov (United States)

    Pfliegler, Walter P; Pusztahelyi, Tünde; Pócsi, István

    2015-07-01

    The application of yeasts has great potential in reducing the economic damage caused by toxigenic fungi in the agriculture. Some yeasts may act as biocontrol agents inhibiting the growth of filamentous fungi. These species may also gain importance in the preservation of agricultural products and in the reduction of their mycotoxin contamination, yet the extent of mycotoxin production in the presence of biocontrol agents is relatively less understood. The application of yeasts in various technological processes may have a direct inhibitory effect on the toxin production of certain molds, which is independent of their growth suppressing effect. Furthermore, several yeast species are capable of accumulating mycotoxins from agricultural products, thereby effectively decontaminating them. Probiotic yeasts or products containing yeast cell wall are also applied to counteract mycotoxicosis in livestock. Several yeast strains are also able to degrade toxins to less-toxic or even non-toxic substances. This intensively researched field would greatly benefit from a deeper knowledge on the genetic and molecular basis of toxin degradation. Moreover, yeasts and their biotechnologically important enzymes may exhibit sensitivity to certain mycotoxins, thereby mounting a considerable problem for the biotechnological industry. It is noted that yeasts are generally regarded as safe; however, there are reports of toxin degrading species that may cause human fungal infections. The aspects of yeast-mycotoxin relations with a brief consideration of strain improvement strategies and genetic modification for improved detoxifying properties and/or mycotoxin resistance are reviewed here.

  19. 中华眼镜蛇心脏毒素(CardiotoxinⅢ)的克隆及其在大肠杆菌和酵母中的表达%Molecular Cloning and Expression of Cardiotoxin Ⅲ from Naja naja atra in E.coli and Yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    陈兴勇; 吕萍; 刘兢; 徐康森

    2008-01-01

    Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60~62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.%心脏毒素cardiotoxinⅢ (CTXⅢ)是中华眼镜蛇毒主要组分之一,根据Genbank中已有的CTXs序列设计特异性引物,从中华眼镜蛇毒中克隆出208bp的CTXⅢ片段,并将该基因片段分别克隆至大肠杆菌His-patch ThioredoxinB载体中,重组CTXⅢ经渗透休克分泌至胞外.将CTXⅢ基因插入酵母pPIC9K分泌型表达载体重组表达,N端带有3个氨基酸(GYT)残基的重组CTXⅢ(rCTXⅢ)分泌量达9.5mg/L,经一步凝胶过滤纯化,其纯度达90%以上,纯化率达65%.经细胞毒性实验检测,纯化的rCTXⅢ 12h的IC50为4.66μg/ml,证实重组蛋白具有良好的生物学活性.

  20. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    Science.gov (United States)

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival. PMID:24305165

  1. Nuclear Import of Yeast Proteasomes

    Directory of Open Access Journals (Sweden)

    Julianne Burcoglu

    2015-08-01

    Full Text Available Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence.

  2. Yeast: A new oil producer?

    Directory of Open Access Journals (Sweden)

    Beopoulos Athanasios

    2012-01-01

    Full Text Available The increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives for the oleochemical field (such as lubricants, adhesives or plastics have created price imbalance in both the alimentary and energy field. Moreover, the lack of non-edible oil feedstock has given rise to concerns on land-use practices and on oil production strategies. Recently, much attention has been paid to the exploitation of microbial oils. Most of them present lipid profiles similar in type and composition to plants and could therefore have many advantages as are no competitive with food, have short process cycles and their cultivation is independent of climate factors. Among microorganisms, yeasts seem to be very promising as they can be easily genetically enhanced, are suitable for large-scale fermentation and are devoid of endotoxins. This review will focus on the recent understanding of yeasts lipid metabolism, the succeeding genetic engineering of the lipid pathways and the recent developments on fermentation techniques that pointed out yeasts as promising alternative producers for oil or plastic.

  3. Distribution of the trehalase activation response and the regulatory trehalase gene among yeast species.

    Science.gov (United States)

    Soto, T; Fernández, J; Cansado, J; Vicente, J; Gacto, M

    1997-12-01

    In Saccharomyces cerevisiae and other yeasts the activity of regulatory trehalases increases in response to the addition of glucose and to thermal changes in the extracellular medium. We have performed an screening on the extent of this response among different representative yeast species and the results show that this ability is displayed only by a few members of the Saccharomycetaceae family. However, all yeasts examined contain a gene related to that coding for regulatory trehalase in S. cerevisiae. This finding reveals that the operational distinction between regulatory and nonregulatory trehalase in yeasts is not a property of the enzyme by itself but relays on the expression of accompanying mechanisms able to modulate trehalase activity.

  4. Yeast Carbon Catabolite Repression†

    Science.gov (United States)

    Gancedo, Juana M.

    1998-01-01

    Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. PMID:9618445

  5. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  6. Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Vinay Kumar Srivastava; Dharani Dhar Dubey

    2007-08-01

    Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.

  7. [Metabolomics analysis of taxadiene producing yeasts].

    Science.gov (United States)

    Yan, Huifang; Ding, Mingzhu; Yuan, Yingjin

    2014-02-01

    In order to study the inherent difference among terpenes producing yeasts from the point of metabolomics, we selected taxadiene producing yeasts as the model system. The changes of cellular metabolites during fermentation log phase of artificial functional yeasts were determined using metabolomics methods. The results represented that compared to W303-1A as a blank control, the metabolites in glycolysis, tricarboxylic acid cycle (TCA) cycle and several amino acids were influenced. And due to the changes of metabolites, the growth of cells was inhibited to a certain extent. Among the metabolites identified, citric acid content in taxadiene producing yeasts changed the most, the decreasing amplitude reached 90% or more. Therefore, citric acid can be a marker metabolite for the future study of artificial functional yeasts. The metabolomics analysis of taxadiene producing yeasts can provide more information in further studies on optimization of terpenes production in heterologous chassis.

  8. Yeasts Diversity in Fermented Foods and Beverages

    Science.gov (United States)

    Tamang, Jyoti Prakash; Fleet, Graham H.

    People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

  9. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast

    Science.gov (United States)

    Li, Yanran; Smolke, Christina D.

    2016-01-01

    Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4′-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery. PMID:27378283

  10. Improved vanillin production in baker's yeast through in silico design

    DEFF Research Database (Denmark)

    Brochado, Ana Rita; Matos, Cláudia; Møller, Birger L.;

    2010-01-01

    Background: Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has...... recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results: Expression of a glycosyltransferase from Arabidopsis...... thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets...

  11. Beer brewing using a fusant between a sake yeast and a brewer's yeast.

    Science.gov (United States)

    Mukai, N; Nishimori, C; Fujishige, I W; Mizuno, A; Takahashi, T; Sato, K

    2001-01-01

    Beer brewing using a fusant between a sake yeast (a lysine auxotrophic mutant of sake yeast K-14) and a brewer's yeast (a respiratory-deficient mutant of the top fermentation yeast NCYC1333) was performed to take advantage of the beneficial characteristics of sake yeasts, i.e., the high productivity of esters, high tolerance to ethanol, and high osmotolerance. The fusant (F-32) obtained was different from the parental yeasts regarding, for example, the assimilation of carbon sources and tolerance to ethanol. A brewing trial with the fusant was carried out using a 100-l pilot-scale plant. The fusant fermented wort more rapidly than the parental brewer's yeast. However, the sedimentation capacity of the fusant was relatively low. The beer brewed using the fusant contained more ethanol and esters compared to that brewed using the parental brewer's yeast. The fusant also obtained osmotolerance in the fermentation of maltose and fermented high-gravity wort well.

  12. Yeast Interacting Proteins Database: YEL005C, YGL079W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available endosome; identified as a transcriptional activator in a high-throughput yeast one-hybrid assay Rows with th...protein localizes to the endosome; identified as a transcriptional activator in a high-throughput yeast one-

  13. Design of Robotic Arm Control System Mimics Human Arm Motion

    Directory of Open Access Journals (Sweden)

    A. Salam Al-Ammri

    2013-01-01

    Full Text Available This paper presents a control system to make the robotic hand mimic human hand motion in real time and offline mode. The human hand tracking system is a wearable sensing arm (potentiometers used to determine the position in space and to sense the grasping task of human hand. The maskable sensing arm was designed with same geometrical arrangement of robotic hand that needs to be controlled. The control software of a robot was implemented using Visual Basic and supported with graphical user interface (GUI. The control algorithm depends on joint to joint mapping method to match between the motions at each joint of portable sensing arm with corresponding joint of a robot in order to make the robot mimic the motion.

  14. The Application of Enzyme and Yeast

    OpenAIRE

    Zhao, Qing

    2012-01-01

    This bachelor’s thesis concerns the application of enzymes and yeasts for bio-industry. The purpose of this work is to understand the basic knowledge about enzyme and yeast, and meanwhile, to find out their different applications. Through comprehensive study, the knowledge was accumulated which brought a clear understanding for the enzyme structure and yeast microorganism, together with their working principles for the bioprocess. For wood-based industry, the different enzymes used in bi...

  15. Revaluation of Waste Yeast from Beer Production

    OpenAIRE

    Nicoleta Suruceanu; Sonia Socaci; Teodora Coldea; Elena Mudura

    2013-01-01

    Brewing yeast is an important waste product from beer production. The valorification of slurry yeast mainly consists of separation of vitamins and important nitrogen compounds. The hops compounds, one of the most important raw materials in beer technology are removed beforehand valorification. The prenylflavonoids compounds from hops are important bioactive compounds that can be revaluation with proper technology. Revaluation of prenylflavonoids from waste yeast into dietary supplement, ident...

  16. NASA's Asteroid Redirect Mission (ARM)

    Science.gov (United States)

    Abell, Paul; Mazanek, Dan; Reeves, David; Naasz, Bo; Cichy, Benjamin

    2015-11-01

    The National Aeronautics and Space Administration (NASA) is developing a robotic mission to visit a large near-Earth asteroid (NEA), collect a multi-ton boulder from its surface, and redirect it into a stable orbit around the Moon. Once returned to cislunar space in the mid-2020s, astronauts will explore the boulder and return to Earth with samples. This Asteroid Redirect Mission (ARM) is part of NASA’s plan to advance the technologies, capabilities, and spaceflight experience needed for a human mission to the Martian system in the 2030s. Subsequent human and robotic missions to the asteroidal material would also be facilitated by its return to cislunar space. Although ARM is primarily a capability demonstration mission (i.e., technologies and associated operations), there exist significant opportunities to advance our knowledge of small bodies in the synergistic areas of science, planetary defense, asteroidal resources and in-situ resource utilization (ISRU), and capability and technology demonstrations. In order to maximize the knowledge return from the mission, NASA is organizing an ARM Investigation Team, which is being preceded by the Formulation Assessment and Support Team. These teams will be comprised of scientists, technologists, and other qualified and interested individuals to help plan the implementation and execution of ARM. An overview of robotic and crewed segments of ARM, including the mission requirements, NEA targets, and mission operations, will be provided along with a discussion of the potential opportunities associated with the mission.

  17. Observations of a Small Sample of Adolescents Experiencing an At-Risk Mental State (ARMS) for Psychosis

    OpenAIRE

    Welsh, Patrick; Tiffin, Paul A.

    2011-01-01

    Some commentaries express concern that the At-Risk Mental State (ARMS) designation can be stigmatizing and induce a lasting sense of personal fragility. However, no studies have actually explored the personal perspectives of those so categorized. The purpose of this study was to explore how adolescents with an ARMS label understand and experience their condition medically and personally. Six participants receiving an ARMS diagnosis were interviewed and the data analyzed using interpretative p...

  18. Heterogeneous expression of osteoclast HsNHA2 in yeast%在酵母中异源表达人破骨细胞钠氢转运蛋白

    Institute of Scientific and Technical Information of China (English)

    黄晓斌; 仲蕾蕾

    2012-01-01

    Objective To heterogeneously express HsNHA2, the potential factor for the differentiation and maturation of human osteoclast, in Saccharomyces cerevisiae, to identify its function to transport Na + as a salt transporter, and to conduct an initial location analysis of its expression in cell. Methods HsNHA2 was amplified using the high fidelity PCR kit. The vector expressed in Saccharomyces cerevisiae was established and transferred into BW31a using electroporation method. The expression in the strain was measured using Western blotting. The growth curve of the strain in high salty concentration medium was observed through NaCl-dependent pressure. The salt-resistant function was observed using phloretin as an inhibitor for HsNHA2. The location of HsNHA2 expressed in the cell was defined using fluorescent in situ staining. Results The results of Western blotting demonstrated that HsNHA2 expressed in BW31a normally. The growth curve showed that BW31a grew in the high-salt concentration medium in the presence of HsNHA2. The growth was inhibited in the 0. 2M NaCl medium when the expression of HsNHA2 was inhibited by phloretin. Immunofluorescence staining demonstrated that HsNHA2 was expressed in the cell membrane. Conclusion HsNHA2 is mainly expressed in the cell membrane to transport Na + as a salt transporter. It can maintain the normal growth of the cells.%目的 在酿酒酵母中异源表达人破骨细胞分化成熟潜在因子钠氢转运蛋白2(HsNHA2),鉴定其作为盐离子载体转运Na+的功能,并对其在细胞中的表达进行初步定位分析.方法 采用高保真PCR试剂盒扩增出HsNHA2;构建酵母表达载体,通过电穿孔的方式转入到酿酒酵母菌株中;Western印迹检测其在酵母菌株的表达;通过NaCl选择压力观察菌株在高盐环境中的生长表型,并用根皮素抑制HsNHA2观察其抗盐功能;最后用细胞原位荧光染色确定HsNHA2在细胞中的表达位置.结果 Western印迹证实HsNHA2在酵

  19. Construction of Killer Wine Yeast Strain

    OpenAIRE

    Seki, Tetsuji; Choi, Eon-Ho; Ryu, Dewey

    1985-01-01

    A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the k...

  20. Scanning ARM Cloud Radar Handbook

    Energy Technology Data Exchange (ETDEWEB)

    Widener, K; Bharadwaj, N; Johnson, K

    2012-06-18

    The scanning ARM cloud radar (SACR) is a polarimetric Doppler radar consisting of three different radar designs based on operating frequency. These are designated as follows: (1) X-band SACR (X-SACR); (2) Ka-band SACR (Ka-SACR); and (3) W-band SACR (W-SACR). There are two SACRs on a single pedestal at each site where SACRs are deployed. The selection of the operating frequencies at each deployed site is predominantly determined by atmospheric attenuation at the site. Because RF attenuation increases with atmospheric water vapor content, ARM's Tropical Western Pacific (TWP) sites use the X-/Ka-band frequency pair. The Southern Great Plains (SGP) and North Slope of Alaska (NSA) sites field the Ka-/W-band frequency pair. One ARM Mobile Facility (AMF1) has a Ka/W-SACR and the other (AMF2) has a X/Ka-SACR.

  1. Specific in vitro toxicity of crude and refined petroleum products: II. Estrogen (alpha and beta) and androgen receptor-mediated responses in yeast assays.

    NARCIS (Netherlands)

    Vrabie, C.M.; Candido, A.; van Duursen, M.B.M.; Jonker, M.T.O.

    2010-01-01

    The present study is the second in a series aiming at a systematic inventory of specific toxic effects of oils. By employing a recombinant yeast stably transfected with human estrogen receptor-alpha (ERalpha) or -beta (ERbeta) or androgen receptor (AR) and expressing yeast enhanced green fluorescent

  2. AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast GRx5 mutant phenotypes and respond to oxidative stress

    Science.gov (United States)

    Arabidopsis monothiol glutaredoxin (Grx), AtGRX4, was targeted to chloroplasts/plastids and had high similarity to yeast Grx5. In yeast expression assays, AtGRX4 localized to the mitochondria and suppressed the sensitivity of grx5 cells to oxidants. In addition, AtGRX4 reduced iron accumulation and ...

  3. Nuclear arms: ethics, strategy, politics

    Energy Technology Data Exchange (ETDEWEB)

    Woolsey, R.J. (ed.)

    1984-01-01

    The range of debate over strategy and arms control today is broader than it was in the late 1950s and early 1960s. In part this is because the early 1980s debate has involved questioning of the fundamental notion of deterrence - by no less than the American Catholic bishops. Today's debate has also seen a national nuclear freeze campaign that, although its congressional supporters have held firmly to a bilateral approach, was tilted perceptibly towards unilateralism by noncongressional leaders at their national conference in February 1983. In one way or another the authors wrestle with different aspects of one central question: could the beginning steps for any consensus be possible, in today's climate, on strategic issues. Ethical issues are addressed in the first 3 papers: Charles Krauthammer, On Nuclear Morality; Patrick Glynn, The Moral Case for the Arms Buildup; and Michael Quinlarc, Thinking Deterrence Through. Three chapters on strategic considerations include: Brent Snowcroft, Understanding the US Strategic Arsenal; William J. Perry, Technological Prospects for US Strategic Forces; and Richard Burt, The Strategic and Political Lessons of INF. Arms control and politics is treated in chapters by: Walter B. Slocombe, Arms Control: Prospects; and Colin S. Gray, Arms Control: Problems. The nonnuclear dimensions of strategy are discussed in chapters by Amory B. and L. Hunter Lovins, Reducing Vulnerability - The Energy Jugular; and Robert Kupperman, Vulnerable America. The chapters on space and defense are: Hans Mark, Arms Control and Space Technology; and Newt Gingrich and John Madison, Space and National Defense. The concluding chapters are by Sen. Sam Nunn, The Need to Reshape Military Stategy; and the editor, R. James Woolsey, The Politics of Vulnerability, 1980-1983.

  4. NetPhosYeast: prediction of protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.;

    2007-01-01

    We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast-an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation...... sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites...... in yeast....

  5. Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis.

    Science.gov (United States)

    Jiménez, Juan J; Borrero, Juan; Diep, Dzung B; Gútiez, Loreto; Nes, Ingolf F; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2013-09-01

    Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

  6. Limitations of yeast surface display in engineering proteins of high thermostability.

    Science.gov (United States)

    Park, Sheldon; Xu, Yao; Stowell, Xiaoran Fu; Gai, Feng; Saven, Jeffery G; Boder, Eric T

    2006-05-01

    Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.

  7. Effect of temperature on replicative aging of the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Molon, Mateusz; Zadrag-Tecza, Renata

    2016-04-01

    The use of the budding yeast Saccharomyces cerevisiae in gerontological studies was based on the assumption that the reproduction limit of a single cell (replicative aging) is a consequence of accumulation of a hypothetical universal "senescence factor" within the mother cell. However, some evidence suggests that molecules or structures proposed as the "aging factor", such as rDNA circles, oxidatively damaged proteins (with carbonyl groups) or mitochondria, have little effect on replicative lifespan of yeast cells. Our results also suggest that protein aggregates associated with Hsp104, treated as a marker of yeast aging, do not seem to affect the numeric value of replicative lifespan of yeast. What these results indicate, however, is the need for finding a different way of expressing age and longevity of yeast cells instead of the commonly used number of daughters produced over units of time, as in the case of other organisms. In this paper, we show that the temperature has a stronger influence on the time of life (the total lifespan) than on the reproductive potential of yeast cells.

  8. 绿色荧光蛋白酿酒酵母表达系统的建立%The Establishment of the Green Fluorescent Protein in Saccharomyces Cerevisiae Yeast Expression System

    Institute of Scientific and Technical Information of China (English)

    祁浩; 刘新利

    2016-01-01

    利用质粒YEplac112为骨架,在其多克隆位点HindIII和EcoRI之间插入水母绿色荧光蛋白( GFP )开放阅读框上下游约1 kb 左右的DNA序列,得到YEplac112-CT质粒,在其多克隆位点 PstI和 BamHI 之间插入750bp左右的GFP DNA序列,构建表达质粒YEplac112-C-GFP-T,以酿酒酵母W303为宿主,通过报告基因GFP验证了所构建的表达载体具有便捷筛选及良好的表达效果。%In this paper,plasmid YEplac112 skeleton was used,green fluorescent protein ( GFP ) was inserted in the multiple cloning sites between HindIII and EcoRI in the open reading frame downstream about 1 KB of DNA sequence, plasmid YEplac112-CT was obtained. 750bp left by the GFP DNA sequence was inserted in the multiple cloning sites PstI and BamHI. Using Saccharomyces cerevisiae W303 as the host, it is proved by reporter gene GFP that the constructed expression vector has good effect,convenient screening and expression.

  9. Yeasts are essential for cocoa bean fermentation.

    Science.gov (United States)

    Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

    2014-03-17

    Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics.

  10. Yeast prions form infectious amyloid inclusion bodies in bacteria

    Directory of Open Access Journals (Sweden)

    Espargaró Alba

    2012-06-01

    Full Text Available Abstract Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM and the Ure2 protein (Ure2p form inclusion bodies (IBs displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.

  11. SREBP controls oxygen-dependent mobilization of retrotransposons in fission yeast.

    Directory of Open Access Journals (Sweden)

    Alfica Sehgal

    2007-08-01

    Full Text Available Retrotransposons are mobile genetic elements that proliferate through an RNA intermediate. Transposons do not encode transcription factors and thus rely on host factors for mRNA expression and survival. Despite information regarding conditions under which elements are upregulated, much remains to be learned about the regulatory mechanisms or factors controlling retrotransposon expression. Here, we report that low oxygen activates the fission yeast Tf2 family of retrotransposons. Sre1, the yeast ortholog of the mammalian membrane-bound transcription factor sterol regulatory element binding protein (SREBP, directly induces the expression and mobilization of Tf2 retrotransposons under low oxygen. Sre1 binds to DNA sequences in the Tf2 long terminal repeat that functions as an oxygen-dependent promoter. We find that Tf2 solo long terminal repeats throughout the genome direct oxygen-dependent expression of adjacent coding and noncoding sequences, providing a potential mechanism for the generation of oxygen-dependent gene expression.

  12. Nuclear physicist, arms control advocate

    CERN Multimedia

    Chang, K

    2002-01-01

    Victor F. Weisskopf, a nuclear physicist who worked on the Manhattan Project to build the first atomic bomb in World War II and later became an ardent advocate of arms control, died Monday at his home in Newton, MA, USA. He was 93 (1 page).

  13. Rhetorical Histories and Arms Negotiations.

    Science.gov (United States)

    Kane, Thomas

    1988-01-01

    Argues that the use of historical events as rhetorical artifacts has sustained cold war assumptions and attitudes; that rhetorical events provide composites for rhetorical histories which become the basis for argumentative appeals; and that these rhetorical histories continue to permeate American diplomacy in general and arms negotiations in…

  14. Ceramide Accumulation in Yeast Yarrowia lipolitica

    Institute of Scientific and Technical Information of China (English)

    周全; 陈国强

    2005-01-01

    Ceramides are a class of lipid molecules widely distributed in eukaryotic cells in small amount. To investigate the possibility of ceramide production by yeast, a yeast strain Yarrowia lipolitica was grown under different conditions including changing carbon/nitrogen ratio, and serine concentration, dissolved oxygen and presence of ethanol. It was found that increased dissolved oxygen supply increased the ceramide content in the yeast 2.5 fold of its normal control level. Ethanol treatment could also enhance ceramide accumulation by 3.3 fold compared with the control although the cell growth was negatively affected. Cellular redox potential was shown to affect ceramide accumulation by the yeast. This was possibly related to the cellular reactive oxygen species presented in the yeast.

  15. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  16. Multiple Identifications in Multi-Armed Bandits

    CERN Document Server

    Bubeck, Sébastien; Viswanathan, Nitin

    2012-01-01

    We study the problem of identifying the top $m$ arms in a multi-armed bandit game. Our proposed solution relies on a new algorithm based on successive rejects of the seemingly bad arms, and successive accepts of the good ones. This algorithmic contribution allows to tackle other multiple identifications settings that were previously out of reach. In particular we show that this idea of successive accepts and rejects applies to the multi-bandit best arm identification problem.

  17. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  18. Haploinsufficiency and the sex chromosomes from yeasts to humans

    Directory of Open Access Journals (Sweden)

    Oliver Stephen G

    2011-02-01

    Full Text Available Abstract Background Haploinsufficient (HI genes are those for which a reduction in copy number in a diploid from two to one results in significantly reduced fitness. Haploinsufficiency is increasingly implicated in human disease, and so predicting this phenotype could provide insights into the genetic mechanisms behind many human diseases, including some cancers. Results In the present work we show that orthologues of Saccharomyces cerevisiae HI genes are preferentially retained across the kingdom Fungi, and that the HI genes of S. cerevisiae can be used to predict haploinsufficiency in humans. Our HI gene predictions confirm known associations between haploinsufficiency and genetic disease, and predict several further disorders in which the phenotype may be relevant. Haploinsufficiency is also clearly relevant to the gene-dosage imbalances inherent in eukaryotic sex-determination systems. In S. cerevisiae, HI genes are over-represented on chromosome III, the chromosome that determines yeast's mating type. This may be a device to select against the loss of one copy of chromosome III from a diploid. We found that orthologues of S. cerevisiae HI genes are also over-represented on the mating-type chromosomes of other yeasts and filamentous fungi. In animals with heterogametic sex determination, accumulation of HI genes on the sex chromosomes would compromise fitness in both sexes, given X chromosome inactivation in females. We found that orthologues of S. cerevisiae HI genes are significantly under-represented on the X chromosomes of mammals and of Caenorhabditis elegans. There is no X inactivation in Drosophila melanogaster (increased expression of X in the male is used instead and, in this species, we found no depletion of orthologues to yeast HI genes on the sex chromosomes. Conclusion A special relationship between HI genes and the sex/mating-type chromosome extends from S. cerevisiae to Homo sapiens, with the microbe being a useful model for

  19. Construction of Yeast Vectors with Resistance to Geneticin

    Institute of Scientific and Technical Information of China (English)

    林会兰; 张广; 周全; 陈国强

    2002-01-01

    Two Escherichia coli-Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin-resistant marker (KanMX4) module coding aminoglycoside 3'-phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.

  20. Breeding of a new wastewater treatment yeast by genetic engineering

    OpenAIRE

    Kato, Miyoshi; Iefuji, Haruyuki

    2011-01-01

    We previously developed a host vector system for the wastewater treatment yeast Hansenula fabianii J640. The promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640 were used for a new expression vector, pHFGE-1. The performance of pHFGE-1 was compared with that of the widely used pG-1 transformant vector. H. fabianii J640 (HF-TAMY) cells were transformed with pHFGE-1, and Saccharomyces cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of whic...

  1. Yeast surface display for screening combinatorial polypeptide libraries.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    1997-06-01

    Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

  2. Expression and Purification of Human Interferon-ω in Yeast Pichia pastoris%人ω干扰素在毕赤酵母中的表达和纯化

    Institute of Scientific and Technical Information of China (English)

    蒙翠凤; 饶晓璐; 郑成已; 王世媛; 陈清西

    2012-01-01

    ω干扰素(IFN-ω)是同IFN-α功能相近但抗原性不同的一类干扰素分子,它具有同IFN-α、IFN-γ以及肿瘤坏死因子α相似的抑制病毒复制的作用.采用5L发酵罐发酵表达IFN-ω,发酵培养液经超滤膜浓缩、SP层析柱梯度分离,得到糖基化的IFN-ω,用基质辅助激光解析电离飞行时间质谱(MALDI TOF-TOF MS)和N端蛋白序列分析仪分析其分子质量和N端序列,用细胞病变抑制法测定活性.结果表明,诱导约48 h时,目的蛋白表达量最高,通过SP梯度洗脱,可将糖基化和非糖基化的IFN-ω分离,质谱分析糖基化蛋白样品,质谱峰为糖基化蛋白特有的峰.纯化后糖基化的IFN-ω抗病毒活性为1.46× 108 U/mL.%IFN-w is a kind of interferon molecules, which has similar function but different antigenicity to IFN-a. It can inhibit virus replication like IFN-a, IFN-y and tumor necrosis factor alpha. 5 L fermenter was used to ferment and interference was expressed. Then,the glycosylated interferon a> was purified by a procedure stepped by ultrafiltration and gradient elution SP chromatography column. Its molecular weight and N amino acid sequence were analyzed by MALDI TOF-TOF MS and the N protein sequence analyzer, respectively. In addition,IFN-a) activity was determined by cell changes suppression method. The results showed that IFN-w expression level reached to highest in induced 48 h. The glycosylated and unglycosylated IFN-co could be separated by SP gradient elution completely. The glycosylated IFN-w showed a series of continuous peaks by mass spectrometry,which is the characteristic peak of glycosyl protein. The antiviral activity of the purified glycosylated IFN-w was 1. 46X108 U/mL

  3. Armed, Non-State Actors and Compliance with International Humanitarian Law

    OpenAIRE

    Furuseth, Astrid

    2014-01-01

    How can we explain compliance with international humanitarian law (IHL) by armed, non-state actors (ANSAs)? All parties to armed conflicts are bound by the IHL. Still, the expectations for rebel group compliance with international law are low, because they are, by definition, political entities fighting against existing authorities. There are, however, ANSAs which express their will to commit or comply with provisions of IHL. This thesis seeks to explore why, and under what conditions, ANSAs ...

  4. Identification of a novel pathway involving a GATA transcription factor in yeast and possibly in plant Zn uptake and homeostasis

    Institute of Scientific and Technical Information of China (English)

    Matthew J. Milner; Nicole S. Pence; Jiping Liu; Leon V. Kochian

    2014-01-01

    To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerules-cens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zap1D. ZAP1 is a transcription factor that activates the Zn dependent transcrip-tion of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRT1 by the plant E2F proteins involves E2F-mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.

  5. Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

    Science.gov (United States)

    De Graeve, Stijn; Marinelli, Sarah; Stolz, Frank; Hendrix, Jelle; Vandamme, Jurgen; Engelborghs, Yves; Van Dijck, Patrick; Thevelein, Johan M

    2013-11-01

    Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

  6. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Stovicek, Vratislav; Borja, Gheorghe M; Forster, Jochen; Borodina, Irina

    2015-11-01

    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.

  7. Repression of a mating type cassette in the fission yeast by four DNA elements

    DEFF Research Database (Denmark)

    Ekwall, K; Nielsen, O; Ruusala, T;

    1991-01-01

    The fission yeast, Schizosaccharomyces pombe, expresses one of two alternative mating types. They are specified by one of two determinants (M or P) present at the mat1 locus. In addition, silent copies of M and P are present on the same chromosome. In the present work we demonstrate that the diff...... partitioning in mitosis to Schizosaccharomyces pombe ars plasmids....

  8. A novel platform for the production of nonhydroxylated gelatins based on the methylotrophic yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Geerlings, Torsten H.; de Boer, Arjo L.; Lunenborg, Marcel G. J.; Veenhuis, Marten; van der Klei, Ida J.

    2007-01-01

    The use of yeast as a host for heterologous expression of proteins that are normally derived from animal tissue is a promising way to ensure defined products that are devoid of potential harmful animal side products. Here we report on the production and secretion of a custom-designed gelatin, Hu3-Hi

  9. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

    NARCIS (Netherlands)

    Klei, I.J. van der; Faber, K.N.; Keizer-Gunnink, I.; Gietl, C.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstr

  10. Growth characteristics of freeze-tolerant baker's yeast Saccharomyces cerevisiae AFY in aerobic batch culture.

    Science.gov (United States)

    Ji, Meng; Miao, Yelian; Chen, Jie Yu; You, Yebing; Liu, Feilong; Xu, Lin

    2016-01-01

    Saccharomyces cerevisiae AFY is a novel baker's yeast strain with strong freeze-tolerance, and can be used for frozen-dough processing. The present study armed to clarify the growth characteristics of the yeast AFY. Aerobic batch culture experiments of yeast AFY were carried out using media with various initial glucose concentrations, and the culture process was analyzed kinetically. The growth of the yeast AFY exhibited a diauxic pattern with the first growth stage consuming glucose and the second growth stage consuming ethanol. The cell yield decreased with increasing initial glucose concentration in the first growth stage, and also decreased with increasing initial ethanol concentration in the second growth stage. In the initial glucose concentration range of 5.0-40.0 g/L, the simultaneous equations of Monod equation, Luedeking-Piret equation and pseudo-Luedeking-Piret equation could be used to describe the concentrations of cell, ethanol and glucose in either of the two exponential growth phases. At the initial glucose concentrations of 5.0, 10.0 and 40.0 g/L, the first exponential growth phase had a maximal specific cell growth rate of 0.52, 0.98 and 0.99 h(-1), while the second exponential growth phase had a maximal specific cell growth rate of 0.11, 0.06 and 0.07 h(-1), respectively. It was indicated that the efficiency of the yeast production could be improved by reducing the ethanol production in the first growth stage. PMID:27186467

  11. A linear discrete dynamic system model for temporal gene interaction and regulatory network influence in response to bioethanol conversion inhibitor HMF for ethanologenic yeast

    Science.gov (United States)

    A linear discrete dynamic system model is constructed to represent the temporal interactions among significantly expressed genes in response to bioethanol conversion inhibitor 5-hydroxymethylfurfural for ethanologenic yeast Saccharomyces cerevisiae. This study identifies the most significant linear...

  12. Modeling competition between yeast strains

    Science.gov (United States)

    de Gee, Maarten; van Mourik, Hilda; de Visser, Arjan; Molenaar, Jaap

    2016-04-01

    We investigate toxin interference competition between S. cerevisiae colonies grown on a solid medium. In vivo experiments show that the outcome of this competition depends strongly on nutrient availability and cell densities. Here we present a new model for S. cerevisiae colonies, calculating the local height and composition of the colonies. The model simulates yeast colonies that show a good fit to experimental data. Simulations of colonies that start out with a homogeneous mixture of toxin producing and toxin sensitive cells can display remarkable pattern formation, depending on the initial ratio of the strains. Simulations in which the toxin producing and toxin sensitive species start at nearby positions clearly show that toxin production is advantageous.

  13. Overview of fission yeast septation.

    Science.gov (United States)

    Pérez, Pilar; Cortés, Juan C G; Martín-García, Rebeca; Ribas, Juan C

    2016-09-01

    Cytokinesis is the final process of the vegetative cycle, which divides a cell into two independent daughter cells once mitosis is completed. In fungi, as in animal cells, cytokinesis requires the formation of a cleavage furrow originated by constriction of an actomyosin ring which is connected to the plasma membrane and causes its invagination. Additionally, because fungal cells have a polysaccharide cell wall outside the plasma membrane, cytokinesis requires the formation of a septum coincident with the membrane ingression. Fission yeast Schizosaccharomyces pombe is a unicellular, rod-shaped fungus that has become a popular model organism for the study of actomyosin ring formation and constriction during cell division. Here we review the current knowledge of the septation and separation processes in this fungus, as well as recent advances in understanding the functional interaction between the transmembrane enzymes that build the septum and the actomyosin ring proteins. PMID:27155541

  14. The wine and beer yeast Dekkera bruxellensis.

    Science.gov (United States)

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. PMID:24932634

  15. 21 CFR 172.590 - Yeast-malt sprout extract.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout... prescribed conditions: (a) The additive is produced by partial hydrolysis of yeast extract (derived...

  16. 21 CFR 172.325 - Bakers yeast protein.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast protein. 172.325 Section 172.325 Food... Special Dietary and Nutritional Additives § 172.325 Bakers yeast protein. Bakers yeast protein may be safely used in food in accordance with the following conditions: (a) Bakers yeast protein is...

  17. 21 CFR 184.1983 - Bakers yeast extract.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b)...

  18. Corning and Kroger turn whey to yeast

    Energy Technology Data Exchange (ETDEWEB)

    1981-11-16

    It is reported that Corning and Kroger intend to build a 35,000 sq. ft. plant in Winchester, Ky., that will turn whey into bakers' yeast. The plant will convert whey from Kroger's dairies into bakers' yeast, supplying about 60% of the yeast needed for nine Kroger bakeries. It will also produce syrups and whey protein concentrate for use in other food processing activities. In addition to making useful products, the project will convert the whey to glucose and galactose. The protein component of the whey will be concentrated and used in various foods and feeds.

  19. Yeast Exocytic v-SNAREs Confer Endocytosis

    OpenAIRE

    Gurunathan, Sangiliyandi; Chapman-Shimshoni, Daphne; Trajkovic, Selena; Gerst, Jeffrey E.

    2000-01-01

    In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1ala43 yeast are d...

  20. Pseudoporphyria associated with consumption of brewers' yeast.

    Science.gov (United States)

    Lim, C K; Rideout, J M; Peters, T J

    1984-06-01

    A case of pseudoporphyria associated with excessive consumption of brewers ' yeast was studied. Detailed analysis of the yeast tablets by high performance liquid chromatography showed the presence of dicarboxylic deuteroporphyrin , mesoporphyrin, and protoporphyrin; coproporphyrin I and III isomers; and uroporphyrin I and III isomers. The faecal porphyrin concentration of the patient taking yeast tablets was significantly increased, resembling the excretion pattern in variegate porphyria. Any patient showing an unusual porphyrin excretion pattern on high performance liquid chromatography should be investigated for a possible dietary cause.

  1. Regenerator cross arm seal assembly

    Science.gov (United States)

    Jackman, Anthony V.

    1988-01-01

    A seal assembly for disposition between a cross arm on a gas turbine engine block and a regenerator disc, the seal assembly including a platform coextensive with the cross arm, a seal and wear layer sealingly and slidingly engaging the regenerator disc, a porous and compliant support layer between the platform and the seal and wear layer porous enough to permit flow of cooling air therethrough and compliant to accommodate relative thermal growth and distortion, a dike between the seal and wear layer and the platform for preventing cross flow through the support layer between engine exhaust and pressurized air passages, and air diversion passages for directing unregenerated pressurized air through the support layer to cool the seal and wear layer and then back into the flow of regenerated pressurized air.

  2. Identification of the transcription factor Znc1p, which regulates the yeast-to-hypha transition in the dimorphic yeast Yarrowia lipolytica.

    Directory of Open Access Journals (Sweden)

    Azul Martinez-Vazquez

    Full Text Available The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica.

  3. Closed-form inverse kinematics for intra-operative mobile C-arm positioning with six degrees of freedom

    Science.gov (United States)

    Wang, Lejing; Zou, Rui; Weidert, Simon; Landes, Juergen; Euler, Ekkehard; Burschka, Darius; Navab, Nassir

    2011-03-01

    For trauma and orthopedic surgery, maneuvering a mobile C-arm X-ray device into a desired position in order to acquire the right picture is a routine task. The precision and ease of use of the C-arm positioning becomes even more important for more advanced imaging techniques as parallax-free X-ray image stitching, for example. Standard mobile C-arms have only five degrees of freedom (DOF), which definitely restricts their motions that have six DOF in 3D Cartesian space. We have proposed a method to model the kinematics of the mobile Carm and operating table as an integrated 6DOF C-arm X-ray imaging system.1 This enables mobile C-arms to be positioned relative to the patient's table with six DOF in 3D Cartesian space. Moving mobile C-arms to a desired position and orientation requires finding the necessary joint values, which is an inverse kinematics problem. In this paper, we present closed-form solutions, i.e. analytic expressions, obtained in an algebraic way for the inverse kinematics problem of the 6DOF C-arm model. In addition, we implement a 6DOF C-arm system for interactively radiation-free C-arm positioning based on a continuous guidance from C-arm pose estimation. For this we employ a visual marker pattern attached under the operating table and a mobile C-arm system augmented by a video camera and mirror construction. In our experiment, repositioning C-arm to a pre-defined pose in a phantom study demonstrates the practicality and accuracy of our developed 6DOF C-arm system.

  4. High power density yeast catalyzed microbial fuel cells

    Science.gov (United States)

    Ganguli, Rahul

    Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density

  5. A heat shock element in the phosphoglycerate kinase gene promoter of yeast.

    OpenAIRE

    Piper, P W; Curran, B; Davies, M W; Hirst, K; Lockheart, A; Ogden, J E; Stanway, C A; Kingsman, A J; Kingsman, S M

    1988-01-01

    The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was ...

  6. YeastWeb: a workset-centric web resource for gene family analysis in yeast

    Directory of Open Access Journals (Sweden)

    Bao Haihua

    2010-07-01

    Full Text Available Abstract Background Currently, a number of yeast genomes with different physiological features have been sequenced and annotated, which provides invaluable information to investigate yeast genetics, evolutionary mechanism, structure and function of gene families. Description YeastWeb is a novel database created to provide access to gene families derived from the available yeast genomes by assigning the genes into putative families. It has many useful features that complement existing databases, such as SGD, CYGD and Génolevures: 1 Detailed computational annotation was conducted with each entry with InterProScan, EMBOSS and functional/pathway databases, such as GO, COG and KEGG; 2 A well established user-friendly environment was created to allow users to retrieve the annotated genes and gene families using functional classification browser, keyword search or similarity-based search; 3 Workset offers users many powerful functions to manage the retrieved data efficiently, associate the individual items easily and save the intermediate results conveniently; 4 A series of comparative genomics and molecular evolution analysis tools are neatly implemented to allow users to view multiple sequence alignments and phylogenetic tree of gene families. At present, YeastWeb holds the gene families clustered from various MCL inflation values from a total of 13 available yeast genomes. Conclusions Given the great interest in yeast research, YeastWeb has the potential to become a useful resource for the scientific community of yeast biologists and related researchers investigating the evolutionary relationship of yeast gene families. YeastWeb is available at http://centre.bioinformatics.zj.cn/Yeast/.

  7. De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae) ▿

    OpenAIRE

    Hansen, Esben H.; Møller, Birger Lindberg; Kock, Gertrud R.; Bünner, Camilla M.; Kristensen, Charlotte; Jensen, Ole R.; Okkels, Finn T.; Olsen, Carl E.; Motawia, Mohammed S.; Hansen, Jørgen

    2009-01-01

    Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast...

  8. The global transcriptional response of fission yeast to hydrogen sulfide.

    Directory of Open Access Journals (Sweden)

    Xu Jia

    Full Text Available BACKGROUND: Hydrogen sulfide (H(2S is a newly identified member of the small family of gasotransmitters that are endogenous gaseous signaling molecules that have a fundamental role in human biology and disease. Although it is a relatively recent discovery and the mechanism of H(2S activity is not completely understood, it is known to be involved in a number of cellular processes; H(2S can affect ion channels, transcription factors and protein kinases in mammals. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have used fission yeast as a model organism to study the global gene expression profile in response to H(2S by microarray. We initially measured the genome-wide transcriptional response of fission yeast to H(2S. Through the functional classification of genes whose expression profile changed in response to H(2S, we found that H(2S mainly influences genes that encode putative or known stress proteins, membrane transporters, cell cycle/meiotic proteins, transcription factors and respiration protein in the mitochondrion. Our analysis showed that there was a significant overlap between the genes affected by H(2S and the stress response. We identified that the target genes of the MAPK pathway respond to H(2S; we also identified that a number of transporters respond to H(2S, these include sugar/carbohydrate transporters, ion transporters, and amino acid transporters. We found many mitochondrial genes to be down regulated upon H(2S treatment and that H(2S can reduce mitochondrial oxygen consumption. CONCLUSION/SIGNIFICANCE: This study identifies potential molecular targets of the signaling molecule H(2S in fission yeast and provides clues about the identity of homologues human proteins and will further the understanding of the cellular role of H(2S in human diseases.

  9. Molecular characterization of a gene that confers 2-deoxyglucose resistance in yeast.

    Science.gov (United States)

    Sanz, P; Randez-Gil, F; Prieto, J A

    1994-09-01

    We have isolated a gene whose expression enables yeast cells to overcome the inhibition of growth produced by the presence of 2-deoxyglucose. The gene contains an open reading frame of 738 bp that may code for a protein of 27,100 Da. Cells carrying this gene contain high levels of a specific 2-deoxyglucose-6-phosphate phosphatase. The expression of this phosphatase is increased by the presence of 2-deoxyglucose and is constant along the growth curve. PMID:7754708

  10. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

    OpenAIRE

    Gurkan Cemal; Ellar David J

    2005-01-01

    Abstract The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promot...

  11. DETERMINATION OF KILLER CHARACTER OF WINE YEAST ISOLATED FROM ISTRA

    OpenAIRE

    Sandi ORLIC; POGAČIĆ, Martina; Ana JEROMEL; Marko KAROGLAN; Kozina, Bernard; IACUMIN, Lucilla; Redžepović, Sulejman

    2008-01-01

    Wild wine yeasts with killer phenotype are widespread in many wine regions of the world. The presence of killer yeasts may become particularly important in wine fermentations conducted by inoculation with selected strains of Saccharomyces cerevisiae. Wild killer yeasts may suppress selected sensitive yeasts inoculated into the must during the fermentation. The goal of this investigation was to identify killer yeast in Istra region using physiological and molecular methods. In total 50 S.cerev...

  12. Enzyme contribution of non-Saccharomyces yeasts to wine production

    OpenAIRE

    Maicas i Prieto, Sergi; Mateo Tolosa, José Juan

    2015-01-01

    The fermentation of grape must to produce wine is a biologically complex process, carried on by yeasts and malolactic bacteria. The yeasts present in spontaneous fermentation may be divided into two groups, the Saccharomyces yeasts, particularly S. cerevisiae, and the non-Saccharomyces yeasts which include members of the genera Rhodotorula, Pichia, Candida, Debaryomyces, Metschtnikowia, Hansenula and Hanseniaspora. S. cerevisiae yeasts are able to convert sugar into ethanol and CO2 via fermen...

  13. Cyanohydrin reactions enhance glycolytic oscillations in yeast

    DEFF Research Database (Denmark)

    Hald, Bjørn Olav; Nielsen, Astrid Gram; Tortzen, Christian;

    2015-01-01

    Synchronous metabolic oscillations can be induced in yeast by addition of glucose and removal of extracellular acetaldehyde (ACAx). Compared to other means of ACAx removal, cyanide robustly induces oscillations, indicating additional cyanide reactions besides ACA to lactonitrile conversion. Here...

  14. Structure and function of yeast alcohol dehydrogenase

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2000-04-01

    Full Text Available 1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer

  15. Structure and function of yeast alcohol dehydrogenase

    OpenAIRE

    VLADIMIR LESKOVAC; SVETLANA TRIVIC

    2000-01-01

    1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer

  16. Genomic Evolution of the Ascomycete Yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Riley, Robert; Haridas, Sajeet; Salamov, Asaf; Boundy-Mills, Kyria; Goker, Markus; Hittinger, Chris; Klenk, Hans-Peter; Lopes, Mariana; Meir-Kolthoff, Jan P.; Rokas, Antonis; Rosa, Carlos; Scheuner, Carmen; Soares, Marco; Stielow, Benjamin; Wisecaver, Jennifer H.; Wolfe, Ken; Blackwell, Meredith; Kurtzman, Cletus; Grigoriev, Igor; Jeffries, Thomas

    2015-03-16

    Yeasts are important for industrial and biotechnological processes and show remarkable metabolic and phylogenetic diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. Phylogenetic analysis of these and previously published yeast genomes helped resolve the placement of species including Saitoella complicata, Babjeviella inositovora, Hyphopichia burtonii, and Metschnikowia bicuspidata. Moreover, we find that alternative nuclear codon usage, where CUG encodes serine instead of leucine, are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes, and a tendency towards more introns in early-diverging species. Analysis of enzyme phylogeny gives insights into the evolution of metabolic capabilities such as methanol utilization and assimilation of alternative carbon sources.

  17. Adenosine triphosphate inhibition of yeast trehalase.

    Science.gov (United States)

    Panek, A D

    1969-09-01

    Yeast trehalase has been found to be inhibited non-competitively by adenosine triphosphate. Such a biological control could explain the accumulation of trehalose during the stationary phase of the growth curve. PMID:5370287

  18. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  19. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    Science.gov (United States)

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast. PMID:26032856

  20. Flavor impacts of glycerol in the processing of yeast fermented beverages: a review.

    Science.gov (United States)

    Zhao, Xiangdong; Procopio, Susanne; Becker, Thomas

    2015-12-01

    Glycerol contributes to the beverage body and fullness. Moreover, it also influences the flavor intensity. As a major byproduct, glycerol not only serves critical roles in yeast osmoregulation and redox balancing, but also acts as the carbon competitor against ethanol in alcoholic fermentation. Therefore, increasing glycerol yield benefits both the flavor and ethanol reduction for the fermented beverages. Glycerol yield has been elevated either by fermentation optimization or by yeast genetic modification. The fermentation optimizations reached maximum 14 g/L glycerol through screening yeast strains and optimizing fermentation parameters. Meanwhile the yeast overexpressing GPD1 (encoding glycerol-3-phosphate dehydrogenase) produced up to 6 folds more glycerol for beer and wine. Except for glycerol improvement, the genetically modified yeasts accumulated dramatically undesirable compounds such as acetaldehyde, acetate and acetoin which are detrimental for beverage flavor. In comparison, the natural high glycerol producers showed strain-specific manner on the yeast-derived aroma compounds like volatile acids, fusel alcohols, esters, and aldehydes. Temperature, sugar concentration, nitrogen composition, oxygen and pH-value, which influence glycerol biosynthesis, also obtained various effects on the production of aromatic compounds. In the current review, we firstly deliberate the organoleptic contributions of glycerol for fermented beverages. Furthermore, glycerol optimization strategies are discussed regarding to the yield improvement, the genes expressions, the overall flavor impacts and the feasibilities in beverage applications. Lastly, for improving beverage flavor by glycerol optimization, a high-throughput platform is proposed to increase the screening capacity of yeast strains and parameters in the processing of fermented beverages. PMID:26604336

  1. alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

    Science.gov (United States)

    Brandis, Katrina A; Holmes, Isaac F; England, Samantha J; Sharma, Nijee; Kukreja, Lokesh; DebBurman, Shubhik K

    2006-01-01

    Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

  2. Metabolic Engineering of Oleaginous Yeasts for Fatty Alcohol Production

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Wei, Hui; Knoshaug, Eric; Van Wychen, Stefanie; Xu, Qi; Himmel, Michael E.; Zhang, Min

    2016-04-25

    To develop pathways for advanced biological upgrading of sugars to hydrocarbons, we are seeking biological approaches to produce high carbon efficiency intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. In this study, we successfully demonstrated fatty alcohol production by oleaginous yeasts Yarrowia lipolytica and Lipomyces starkeyi by expressing a bacteria-derived fatty acyl-CoA reductase (FAR). Moreover, we find higher extracellular distribution of fatty alcohols produced by FAR-expressing L. starkeyi strain as compared to Y. lipolytica strain, which would benefit the downstream product recovery process. In both oleaginous yeasts, long chain length saturated fatty alcohols were predominant, accounting for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Taken together, our work demonstrates that in addition to Y. lipolytica, L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering. We believe strain and process development both will significantly contribute to our goal of producing scalable and cost-effective fatty alcohols from renewable biomass.

  3. Physiological and environmental control of yeast prions

    OpenAIRE

    Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

    2013-01-01

    Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion ...

  4. Production of biopharmaceutical proteins by yeast

    OpenAIRE

    Nielsen, Jens

    2012-01-01

    Production of recombinant proteins for use as pharmaceuticals, so-called biopharmaceuticals, is a multi-billion dollar industry. Many different cell factories are used for the production of biopharmaceuticals, but the yeast Saccharomyces cerevisiae is an important cell factory as it is used for production of several large volume products. Insulin and insulin analogs are by far the dominating biopharmaceuticals produced by yeast, and this will increase as the global insulin market is expected ...

  5. Determination of tritium in wine yeast samples

    International Nuclear Information System (INIS)

    Analytical procedures were developed to determine tritium in wine and wine yeast samples. The content of organic compounds affecting the LSC measurement is reduced by fractioning distillation for wine samples and azeotropic distillation/fractional distillation for wine yeast samples. Finally, the water samples were normally distilled with K MO4. The established procedures were successfully applied for wine and wine samples from Murfatlar harvests of the years 1995 and 1996. (authors)

  6. The wine and beer yeast Dekkera bruxellensis

    OpenAIRE

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P.; Piškur, Jure

    2014-01-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beer...

  7. OPTIMIZATION OF YEAST FOR ETHANOL PRODUCTION

    OpenAIRE

    Taghizadeh Ghassem; Delbari Azam Sadat; Kulkarni D. K.

    2012-01-01

    The production of pure ethanol apparently begins in the 12-14th century. Improvements in the distillation process with the condensation of vapors of lower boiling liquids. Ethanol is produced commercially by chemical synthesis or biosynthesis. High ethanol producing yeast exhibits rapid metabolic activity and a high fermentation rate with high product output in less time.Yeasts were isolated from Corn, Curd, Grapes, Water 1, Water 2, and Paneer. Isolation was done on MGYP (Malt Extract Glucos...

  8. Stationary phase in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    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 c...

  9. Mapping the functional yeast ABC transporter interactome

    OpenAIRE

    Snider, Jamie; Hanif, Asad; Lee, Mid Eum; Jin, Ke; Yu, Analyn R.; Graham, Chris; Chuk, Matthew; Damjanovic, Dunja; Wierzbicka, Marta; Tang, Priscilla; Balderes, Dina; Wong, Victoria; Jessulat, Matthew; Darowski, Katelyn D.; Luis, Bryan-Joseph San

    2013-01-01

    ABC transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used Membrane Yeast Two-Hybrid (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, and combined this data with previously reported yeast ABC transporter interactions in the BioGRID databa...

  10. Principles of chromosomal organization: lessons from yeast

    OpenAIRE

    Zimmer, Christophe; Fabre, Emmanuelle

    2011-01-01

    The spatial organization of genes and chromosomes plays an important role in the regulation of several DNA processes. However, the principles and forces underlying this nonrandom organization are mostly unknown. Despite its small dimension, and thanks to new imaging and biochemical techniques, studies of the budding yeast nucleus have led to significant insights into chromosome arrangement and dynamics. The dynamic organization of the yeast genome during interphase argues for both the physica...

  11. Multidrug resistant yeasts in synanthropic wild birds

    Directory of Open Access Journals (Sweden)

    Somanath Sushela

    2010-03-01

    Full Text Available Abstract Background The aim of this study was to investigate the presence of multidrug resistant yeasts in the faeces of synanthropic wild birds from the Bangsar suburb of Kuala Lumpur. Methods Species characterisations of yeast isolates and determinations of antimycotic susceptibility profiles were undertaken using the commercial characterization kit, Integral System Yeasts Plus (Liofilchem, Italy. Results Fourteen species of yeasts were detected in the bird faecal samples.Candida albicans was present in 28.89% of bird faecal samples, Candida krusei (13.33%, Candida tropicalis (4.44%, Candida glabrata (4.44%, Candida parapsilosis (2.22%, Candida lambica (2.22%, Candida stellatoidea (2.22%, Candida rugosa (2.22% and Candida lusitaniae (2.22%. Amongst the non-candidal yeast isolates, Cryptococcus laurentii was present in 6.67% of bird faecal samples, Cryptococcus uniguttulatus (4.44%, Saccharomyces cerevisiae (4.44%, Trichosporon pullulans (2.22%, Trichosporon pullulans/Cryptococcus albidus (8.89% and Rhodotorula rubra/Rhodotorula glutinis (4.44%. Of the isolated yeasts, 18.1% (or 26/144 were found to be resistant to all 11 antimycotic agents they were tested against i.e. Nystatin, Amphotericin B, Flucytosine, Econazole, Ketoconazole, Clotrimazole, Miconazole, Itraconazole, Voriconazole, Fluconazole 16 and Fluconazole 64. 45.8% (or 66/144 of the bird faecal yeast isolates were resistant to four or more of the 11 antimycotic agents they were tested against. Conclusions This finding is of public health significance as these synanthropic wild birds may be reservoirs for transmission of drug resistant yeast infections to humans.

  12. Uniform yeast cell assembly via microfluidics

    OpenAIRE

    Chang, Ya-Wen; He, Peng; Marquez, Samantha M.; Cheng, Zhengdong

    2012-01-01

    This paper reports the use of microfluidic approaches for the fabrication of yeastosomes (yeast-celloidosomes) based on self-assembly of yeast cells onto liquid-solid or liquid-gas interfaces. Precise control over fluidic flows in droplet- and bubble-forming microfluidic devices allows production of monodispersed, size-selected templates. The general strategy to organize and assemble living cells is to tune electrostatic attractions between the template (gel or gas core) and the cells via sur...

  13. A new biological test of water toxicity-yeast Saccharomyces cerevisiae conductometric test.

    Science.gov (United States)

    Dolezalova, Jaroslava; Rumlova, Lubomira

    2014-11-01

    This new biological test of water toxicity is based on monitoring of specific conductivity changes of yeast Saccharomyces cerevisiae suspension as a result of yeast fermentation activity inhibition in toxic conditions. The test was verified on ten substances with various mechanisms of toxic effect and the results were compared with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminescence inhibition (EN ISO 11348-2) and with the results of the S. cerevisiae lethal test (Rumlova and Dolezalova, 2012). The new biological test - S. cerevisiae conductometric test - is an express method developed primarily for field conditions. It is applicable in case of need of immediate information about water toxicity. Fast completion is an advantage of this test (time necessary for test completion is about 60min), the test is simple and the test organism - dried instant yeast - belongs among its biggest advantages because of its long-term storage life and broad availability.

  14. Yeast profilin complements profilin deficiency in transgenic tomato fruits and allows development of hypoallergenic tomato fruits.

    Science.gov (United States)

    Le, Lien Q; Mahler, Vera; Scheurer, Stephan; Foetisch, Kay; Braun, Yvonne; Weigand, Daniela; Enrique, Ernesto; Lidholm, Jonas; Paulus, Kathrin E; Sonnewald, Sophia; Vieths, Stefan; Sonnewald, Uwe

    2010-12-01

    Gene silencing of Lyc e 1 leads to reduced allergenicity of tomato fruits but impaired growth of transgenic tomato plants. The aim of the study was to restore growth of Lyc e 1-deficient tomato plants while retaining reduced allergenicity by simultaneous complementation of profilin deficiency by expression of nonallergenic yeast profilin. Transgenic plants were generated and tested by RT-PCR and immunoblotting; allergenicity of yeast profilin and transgenic fruits was investigated by IgE binding, basophil activation, and skin-prick tests. Lyc e 1 content of transgenic tomato fruits was foods with reduced allergen accumulation, can be generated even if the allergen fulfills an essential cellular function in the plant. In summary, our findings of efficiently complementing profilin-deficient tomato plants by coexpression of low allergenic yeast profilin demonstrate the feasibility of creating low-allergenic food even if the allergen fulfills essential cellular functions.

  15. A yeast surface display system for the discovery of ligands that trigger cell activation.

    Science.gov (United States)

    Cho, B K; Kieke, M C; Boder, E T; Wittrup, K D; Kranz, D M

    1998-11-01

    Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

  16. Revaluation of Waste Yeast from Beer Production

    Directory of Open Access Journals (Sweden)

    Nicoleta Suruceanu

    2013-11-01

    Full Text Available Brewing yeast is an important waste product from beer production. The valorification of slurry yeast mainly consists of separation of vitamins and important nitrogen compounds. The hops compounds, one of the most important raw materials in beer technology are removed beforehand valorification. The prenylflavonoids compounds from hops are important bioactive compounds that can be revaluation with proper technology. Revaluation of prenylflavonoids from waste yeast into dietary supplement, identification and quantification of xanthohumol by HPLC method. Waste yeast from brewery pilot plant of USAMV Cluj Napoca it was dried by atomization and the powder was analyzed on xanthohumol content by HPLC method. For quantification a calibration curve it was used. The process of drying by atomisation lead to a powder product. It was used malt dextrin powder for stabilisation. The final product it was encapsulated. The xanthohumol content of powdered yeast it was 1.94 µg/ml. In conclusion the slurry yeast from beer production it is an important source of prenylflavonoids compounds.

  17. Flor Yeast: New Perspectives Beyond Wine Aging.

    Science.gov (United States)

    Legras, Jean-Luc; Moreno-Garcia, Jaime; Zara, Severino; Zara, Giacomo; Garcia-Martinez, Teresa; Mauricio, Juan C; Mannazzu, Ilaria; Coi, Anna L; Bou Zeidan, Marc; Dequin, Sylvie; Moreno, Juan; Budroni, Marilena

    2016-01-01

    The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air-liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed. PMID:27148192

  18. Flor Yeast: New Perspectives Beyond Wine Aging

    Science.gov (United States)

    Legras, Jean-Luc; Moreno-Garcia, Jaime; Zara, Severino; Zara, Giacomo; Garcia-Martinez, Teresa; Mauricio, Juan C.; Mannazzu, Ilaria; Coi, Anna L.; Bou Zeidan, Marc; Dequin, Sylvie; Moreno, Juan; Budroni, Marilena

    2016-01-01

    The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed. PMID:27148192

  19. Modeling diauxic glycolytic oscillations in yeast

    DEFF Research Database (Denmark)

    Hald, Bjørn Olav; Sørensen, Preben Graae

    2010-01-01

    Glycolytic oscillations in a stirred suspension of starved yeast cells is an excellent model system for studying the dynamics of metabolic switching in living systems. In an open-flow system the oscillations can be maintained indefinitely at a constant operating point where they can be characteri......Glycolytic oscillations in a stirred suspension of starved yeast cells is an excellent model system for studying the dynamics of metabolic switching in living systems. In an open-flow system the oscillations can be maintained indefinitely at a constant operating point where they can...... for investigations of central metabolism dynamics of yeast cells. We have previously proposed a model for the open system comprised of the primary fermentative reactions in yeast that quantitatively describes the oscillatory dynamics. However, this model fails to describe the transient behavior of metabolic...... switching in a closed-system experiment by feeding the yeast suspension with a glucose pulse-notably the initial NADH spike and final NADH rise. Another object of this study is to gain insight into the secondary low-flux metabolic pathways by feeding starved yeast cells with various metabolites...

  20. [Construction and Identification of the Bait Vector Containing Duck Circovirus Cap Gene for the Yeast Two-hybrid System].

    Science.gov (United States)

    Xu, Yu; Zhang, Zhilong; Lu, Yanyan; Zhang, Lei; Li, Pengfei; Jia, Renyong

    2015-05-01

    To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system, the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR, restriction enzyme digestion, and sequence analysis. After transformation into a Y2HGold yeast strain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected using different dropout minimal base. PCR reaction, restriction enzyme digestion, and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pG- BKT7. Western blotting showed that the whole Cap protein was expressed. The recombinant bait protein had no toxicity and self-activation. Therefore, the bait vector with the Cap gene was constructed successfully, providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.

  1. Effects of wort gravity and nitrogen level on fermentation performance of brewer's yeast and the formation of flavor volatiles.

    Science.gov (United States)

    Lei, Hongjie; Zhao, Haifeng; Yu, Zhimin; Zhao, Mouming

    2012-03-01

    Normal gravity wort and high gravity wort with different nitrogen levels were used to examine their effects on the fermentation performance of brewer's yeast and the formation of flavor volatiles. Results showed that both the wort gravity and nitrogen level had significant impacts on the growth rate, viability, flocculation, and gene expression of brewer's yeast and the levels of flavor volatiles. The sugar (glucose, maltose, and maltotriose) consumption rates and net cell growth decreased when high gravity worts were used, while these increased with increasing nitrogen level. Moreover, high gravity resulted in lower expression levels of ATF1, BAP2, BAT1, HSP12, and TDH, whereas the higher nitrogen level caused higher expression levels for these genes. Furthermore, the lower nitrogen level resulted in increases in the levels of higher alcohols and esters at high wort gravity. All these results demonstrated that yeast physiology and flavor balance during beer brewing were significantly affected by the wort gravity and nitrogen level.

  2. More than just trash bins? Potential roles for extracellular vesicles in the vertical and horizontal transmission of yeast prions.

    Science.gov (United States)

    Kabani, Mehdi; Melki, Ronald

    2016-05-01

    In the yeast Saccharomyces cerevisiae, an ensemble of structurally and functionally diverse cytoplasmic proteins has the ability to form self-perpetuating protein aggregates (e.g. prions) which are the vectors of heritable non-Mendelian phenotypic traits. Whether harboring these prions is deleterious-akin to mammalian degenerative disorders-or beneficial-as epigenetic modifiers of gene expression-for yeasts has been intensely debated and strong arguments were made in support of both views. We recently reported that the yeast prion protein Sup35p is exported via extracellular vesicles (EV), both in its soluble and aggregated infectious states. Herein, we discuss the possible implications of this observation and propose several hypotheses regarding the roles of EV in both vertical and horizontal propagation of 'good' and 'bad' yeast prions. PMID:26553335

  3. Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast

    DEFF Research Database (Denmark)

    Kleinow, T.; Bhalerao, R.; Breuer, F.;

    2000-01-01

    Yeast Snf4 is a prototype of activating gamma-subunits of conserved Snf1/AMPK-related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast....... By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc...

  4. The Asteroid Redirect Mission (ARM)

    Science.gov (United States)

    Abell, Paul; Gates, Michele; Johnson, Lindley; Chodas, Paul; Mazanek, Dan; Reeves, David; Ticker, Ronald

    2016-07-01

    To achieve its long-term goal of sending humans to Mars, the National Aeronautics and Space Administration (NASA) plans to proceed in a series of incrementally more complex human spaceflight missions. Today, human flight experience extends only to Low-Earth Orbit (LEO), and should problems arise during a mission, the crew can return to Earth in a matter of minutes to hours. The next logical step for human spaceflight is to gain flight experience in the vicinity of the Moon. These cis-lunar missions provide a "proving ground" for the testing of systems and operations while still accommodating an emergency return path to the Earth that would last only several days. Cis-lunar mission experience will be essential for more ambitious human missions beyond the Earth-Moon system, which will require weeks, months, or even years of transit time. In addition, NASA has been given a Grand Challenge to find all asteroid threats to human populations and know what to do about them. Obtaining knowledge of asteroid physical properties combined with performing technology demonstrations for planetary defense provide much needed information to address the issue of future asteroid impacts on Earth. Hence the combined objectives of human exploration and planetary defense give a rationale for the Asteroid Re-direct Mission (ARM). Mission Description: NASA's ARM consists of two mission segments: 1) the Asteroid Redirect Robotic Mission (ARRM), the first robotic mission to visit a large (greater than ~100 m diameter) near-Earth asteroid (NEA), collect a multi-ton boulder from its surface along with regolith samples, demonstrate a planetary defense technique, and return the asteroidal material to a stable orbit around the Moon; and 2) the Asteroid Redirect Crewed Mission (ARCM), in which astronauts will take the Orion capsule to rendezvous and dock with the robotic vehicle, conduct multiple extravehicular activities to explore the boulder, and return to Earth with samples. NASA's proposed

  5. Purification and crystallization of the yeast translation elongation factor eEF3

    DEFF Research Database (Denmark)

    Andersen, Christian Folsted; Anand, Monika; Boesen, Thomas;

    2004-01-01

    A Saccharomyces cerevisiae strain expressing full-length histidine-tagged translation elongation factor 3 (eEF3) as the only form of the protein facilitated purification of the factor for both structural and functional studies. Additionally, an identical full-length form has been successfully...... expressed in Escherichia coli and a C-terminally truncated form of histidine-tagged eEF3 has been successfully expressed in E. coli and S. cerevisiae. Both forms have been crystallized and crystals of the truncated protein expressed in yeast diffract synchrotron radiation to a maximum resolution of 2.3 A...

  6. A systems-level approach for metabolic engineering of yeast cell factories.

    Science.gov (United States)

    Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

    2012-03-01

    The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories. PMID:22188344

  7. An improved, bias-reduced probabilistic functional gene network of baker's yeast, Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Insuk Lee

    Full Text Available BACKGROUND: Probabilistic functional gene networks are powerful theoretical frameworks for integrating heterogeneous functional genomics and proteomics data into objective models of cellular systems. Such networks provide syntheses of millions of discrete experimental observations, spanning DNA microarray experiments, physical protein interactions, genetic interactions, and comparative genomics; the resulting networks can then be easily applied to generate testable hypotheses regarding specific gene functions and associations. METHODOLOGY/PRINCIPAL FINDINGS: We report a significantly improved version (v. 2 of a probabilistic functional gene network of the baker's yeast, Saccharomyces cerevisiae. We describe our optimization methods and illustrate their effects in three major areas: the reduction of functional bias in network training reference sets, the application of a probabilistic model for calculating confidences in pair-wise protein physical or genetic interactions, and the introduction of simple thresholds that eliminate many false positive mRNA co-expression relationships. Using the network, we predict and experimentally verify the function of the yeast RNA binding protein Puf6 in 60S ribosomal subunit biogenesis. CONCLUSIONS/SIGNIFICANCE: YeastNet v. 2, constructed using these optimizations together with additional data, shows significant reduction in bias and improvements in precision and recall, in total covering 102,803 linkages among 5,483 yeast proteins (95% of the validated proteome. YeastNet is available from http://www.yeastnet.org.

  8. Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

    Science.gov (United States)

    Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

    2016-09-01

    Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %). PMID:27469174

  9. Signaling of human frizzled receptors to the mating pathway in yeast.

    Directory of Open Access Journals (Sweden)

    Dietmar Dirnberger

    Full Text Available Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs. The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2 can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.

  10. Signaling of human frizzled receptors to the mating pathway in yeast.

    Science.gov (United States)

    Dirnberger, Dietmar; Seuwen, Klaus

    2007-09-26

    Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.

  11. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference.

    Science.gov (United States)

    Murphy, Katherine A; Tabuloc, Christine A; Cervantes, Kevin R; Chiu, Joanna C

    2016-01-01

    RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally found growing on the surface of fruit crops, constitutes a major component of the Drosophila microbiome, and is highly attractive to Drosophila. Thus, this naturally attractive yeast biopesticide can deliver dsRNA to an insect pest without the need for genetic crop modification. We demonstrate that this biopesticide decreases larval survivorship, and reduces locomotor activity and reproductive fitness in adults, which are indicative of general health decline. To our knowledge, this is the first study to show that yeast can be used to deliver dsRNA to an insect pest. PMID:26931800

  12. Isolation, identification, and activity in vitro of killer yeasts against Colletotrichum gloeosporioides isolated from tropical fruits.

    Science.gov (United States)

    de Lima, Jaqueline Rabelo; Gonçalves, Luciana Rocha Barros; Brandão, Luciana Rocha; Rosa, Carlos Augusto; Viana, Francisco Marto Pinto

    2013-07-01

    A total of 580 yeasts strains, isolated from Ceara State of Brasil, were evaluated for their ability to produce killer toxin. Of these strains, 29 tested positive for the killer phenotype and were further evaluated for their ability to control Colletotrichum gloeosporioides germination in vitro. All yeast strains that expressed the killer phenotype were characterized by sequencing the D1/D2 regions of the large subunit of the rRNA gene. Five yeast strains provided a significant reduction in mycelial growth and conidial germination of C. gloeosporioides in vitro, especially Meyerozyma guilliermondii, which was able to reduce the fungal mycelial growth on solid medium (potato dextrose agar (PDA)) by 60% and block 100% of conidia germination in liquid media (potato dextrose broth (PDB)). Filtering and autoclaving the liquid cultures had no effect on the growth of the pathogen. These results indicate the potential use of antagonist yeasts isolated from tropical fruits in the control of anthracnose caused by C. gloeosporioides in papaya. Further elucidation of main mechanisms involved on anthracnose control by these yeasts could be helpful for the development of biocontrol techniques related to the management of this disease in tropical fruits. PMID:22915228

  13. Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications.

    Science.gov (United States)

    Takagi, Hiroshi

    2008-11-01

    Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T (m) of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive gamma-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production. PMID:18802692

  14. Virulence markers of opportunistic black yeast in Exophiala.

    Science.gov (United States)

    Sav, Hafize; Ozakkas, Fatma; Altınbas, Rabiye; Kiraz, Nuri; Tümgör, Ayşegül; Gümral, Ramazan; Döğen, Aylin; Ilkit, Macit; de Hoog, G Sybren

    2016-06-01

    The black yeast genus Exophiala is known to cause a wide variety of diseases in severely ill individuals but can also affect immunocompetent individuals. Virulence markers and other physiological parameters were tested in eight clinical and 218 environmental strains, with a specific focus on human-dominated habitats for the latter. Urease and catalase were consistently present in all samples; four strains expressed proteinase and three strains expressed DNase, whereas none of the strains showed phospholipase, haemolysis, or co-haemolysis activities. Biofilm formation was identified in 30 (13.8%) of the environmental isolates, particularly in strains from dishwashers, and was noted in only two (25%) of the clinical strains. These results indicate that virulence factors are inconsistently present in the investigated Exophiala species, suggesting opportunism rather than pathogenicity. PMID:26857806

  15. Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis

    Institute of Scientific and Technical Information of China (English)

    GUO Li-qiong; LIU Er-xian; WANG Jie; LIN Jun-fang

    2009-01-01

    Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 (containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B) and plasmid pLg-hph (containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph) were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL-1 hygromycin. The putative transformantswere obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation.Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub-culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.

  16. Deciphering human heat shock transcription factor 1 regulation via post-translational modification in yeast.

    Directory of Open Access Journals (Sweden)

    Liliana Batista-Nascimento

    Full Text Available Heat shock transcription factor 1 (HSF1 plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications.

  17. Study on a 7-DOF anthropomorphic weld arm

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A kind of new obstacle space expression is proposed in this paper, in which a virtual force field (VFF) is built. Using the torque and joint optimization acted by the virtual force field on the anthropomorphic weld arm, the real-time selection of a redundant join (R-joint) is done and its equivalent virtual torque is obtained, thus the redundant joint can be controlled with whose force feedback. An inverse kinematics solution of a 6-DOF robot is applied to other six joints of the arm. Simulation experiments indicate the new inverse kinematics solution has perfect collision avoidance effect, and it is well simplified. Therefore, it can be applied to a welding task in complex operation space.

  18. Assembly factors for the membrane arm of human complex I

    Science.gov (United States)

    Andrews, Byron; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2013-01-01

    Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron–sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I. PMID:24191001

  19. Introduction to Reading and Visualizing ARM Data

    Energy Technology Data Exchange (ETDEWEB)

    Mather, James [Pacific Northwest National Laboratory

    2014-02-18

    Atmospheric Radiation Measurement (ARM) Program standard data format is NetCDF 3 (Network Common Data Form). The object of this tutorial is to provide a basic introduction to NetCDF with an emphasis on aspects of the ARM application of NetCDF. The goal is to provide basic instructions for reading and visualizing ARM NetCDF data with the expectation that these examples can then be applied to more complex applications.

  20. Lever arm dysfunction in cerebral palsy gait

    OpenAIRE

    Theologis, Tim

    2013-01-01

    Skeletal structures act as lever arms during walking. Muscle activity and the ground reaction against gravity exert forces on the skeleton, which generate torque (moments) around joints. These lead to the sequence of movements which form normal human gait. Skeletal deformities in cerebral palsy (CP) affect the function of bones as lever arms and compromise gait. Lever arm dysfunction should be carefully considered when contemplating treatment to improve gait in children with CP.