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Sample records for diverse yeast genes

  1. The yeast SEN1 gene is required for the processing of diverse RNA classes.

    PubMed

    Ursic, D; Himmel, K L; Gurley, K A; Webb, F; Culbertson, M R

    1997-12-01

    A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.

  2. RPD1 (SIN3/UME4) is required for maximal activation and repression of diverse yeast genes

    SciTech Connect

    Vidal, M.; Gaber, R.F. ); Strich, R.; Esposito, R.E. )

    1991-12-01

    The authors show that the extent of transcriptional regulation of many, apparently unrelated, genes in Saccharomyces cerevisiae is dependent on RPD1 (and RPD3). Genes regulated by stimuli as diverse as external signals (PH05), cell differentiation processes (SPO11 and SPO13), cell type (RME1, FUS1, H0, TY2, STE6, STE3, and BAR1), and genes whose regulatory signals remain unknown (TRK2) depend on RPD1 to achieve maximal states of transcriptional regulation. RPD1 enhances both positive and negative regulation of these genes: in rpdl{Delta} mutants, higher levels of expression are observed under repression conditions and lower levels are observed under activation conditions. They show that several independent genetic screens, designed to identify yeast transcriptional regulator, have detected the RPD1 locus (also known as SIN3, SD11, and UME4). The inferred RPD1 protein contains four regions predicted to take on helix-loop-helix-like secondary structures and three regions (acidic, glutamine rich, and proline rich) reminiscent of the activating domains of transcriptional activators.

  3. Yeast and yeast-like diversity in the southernmost glacier of Europe (Calderone Glacier, Apennines, Italy).

    PubMed

    Branda, Eva; Turchetti, Benedetta; Diolaiuti, Guglielmina; Pecci, Massimo; Smiraglia, Claudio; Buzzini, Pietro

    2010-06-01

    The present study reports the characterization of psychrophilic yeast and yeast-like diversity in cold habitats (superficial and deep sediments, ice cores and meltwaters) of the Calderone Glacier (Italy), which is the southernmost glacier in Europe. After incubation at 4 and 20 degrees C, sediments contained about 10(2)-10(3) CFU of yeasts g(-1). The number of viable yeast cells in ice and meltwaters was several orders of magnitude lower. The concomitant presence of viable bacteria and filamentous fungi has also been observed. In all, 257 yeast strains were isolated and identified by 26S rRNA gene D1/D2 and internal transcribed spacers (1 and 2) sequencing as belonging to 28 ascomycetous and basidiomycetous species of 11 genera (Candida, Cystofilobasidium, Cryptococcus, Dioszegia, Erythrobasidium, Guehomyces, Mastigobasidium, Mrakia, Mrakiella, Rhodotorula and Sporobolomyces). Among them, the species Cryptococcus gastricus accounted for almost 40% of the total isolates. In addition, 12 strains were identified as belonging to the yeast-like species Aureobasidium pullulans and Exophiala dermatitidis, whereas 15 strains, presumably belonging to new species, yet to be described, were also isolated. Results herein reported indicate that the Calderone Glacier, although currently considered a vanishing ice body due to the ongoing global-warming phenomenon, still harbors viable psychrophilic yeast populations. Differences of yeast and yeast-like diversity between the glacier under study and other worldwide cold habitats are also discussed.

  4. Genome Diversity and Evolution in the Budding Yeasts (Saccharomycotina)

    PubMed Central

    Dujon, Bernard A.; Louis, Edward J.

    2017-01-01

    Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. PMID:28592505

  5. Diversity and adaptive evolution of Saccharomyces wine yeast: a review

    PubMed Central

    Marsit, Souhir; Dequin, Sylvie

    2015-01-01

    Saccharomyces cerevisiae and related species, the main workhorses of wine fermentation, have been exposed to stressful conditions for millennia, potentially resulting in adaptive differentiation. As a result, wine yeasts have recently attracted considerable interest for studying the evolutionary effects of domestication. The widespread use of whole-genome sequencing during the last decade has provided new insights into the biodiversity, population structure, phylogeography and evolutionary history of wine yeasts. Comparisons between S. cerevisiae isolates from various origins have indicated that a variety of mechanisms, including heterozygosity, nucleotide and structural variations, introgressions, horizontal gene transfer and hybridization, contribute to the genetic and phenotypic diversity of S. cerevisiae. This review will summarize the current knowledge on the diversity and evolutionary history of wine yeasts, focusing on the domestication fingerprints identified in these strains. PMID:26205244

  6. Genome Diversity and Evolution in the Budding Yeasts (Saccharomycotina).

    PubMed

    Dujon, Bernard A; Louis, Edward J

    2017-06-01

    Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. Copyright © 2017 by the Genetics Society of America.

  7. Functional Diversity of Silencers in Budding Yeasts

    PubMed Central

    Sjöstrand, Jimmy O. O.; Kegel, Andreas; Åström, Stefan U.

    2002-01-01

    We studied the silencing of the cryptic mating-type loci HMLα and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLα. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HMLα1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces. PMID:12456003

  8. Cell cycle regulated gene expression in yeasts.

    PubMed

    McInerny, Christopher J

    2011-01-01

    The regulation of gene expression through the mitotic cell cycle, so that genes are transcribed at particular cell cycle times, is widespread among eukaryotes. In some cases, it appears to be important for control mechanisms, as deregulated expression results in uncontrolled cell divisions, which can cause cell death, disease, and malignancy. In this review, I describe the current understanding of such regulated gene expression in two established simple eukaryotic model organisms, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In these two yeasts, the global pattern of cell cycle gene expression has been well described, and most of the transcription factors that control the various waves of gene expression, and how they are in turn themselves regulated, have been characterized. As related mechanisms occur in all other eukaryotes, including humans, yeasts offer an excellent paradigm to understand this important molecular process. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Yeasts Diversity in Fermented Foods and Beverages

    NASA Astrophysics Data System (ADS)

    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

  10. Improving industrial yeast strains: exploiting natural and artificial diversity

    PubMed Central

    Steensels, Jan; Snoek, Tim; Meersman, Esther; Nicolino, Martina Picca; Voordeckers, Karin; Verstrepen, Kevin J

    2014-01-01

    Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as ‘global transcription machinery engineering’ (gTME), to induce genetic variation, providing a new source of yeast genetic diversity. PMID:24724938

  11. Improving industrial yeast strains: exploiting natural and artificial diversity.

    PubMed

    Steensels, Jan; Snoek, Tim; Meersman, Esther; Picca Nicolino, Martina; Voordeckers, Karin; Verstrepen, Kevin J

    2014-09-01

    Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as 'global transcription machinery engineering' (gTME), to induce genetic variation, providing a new source of yeast genetic diversity. © 2014 The Authors. FEMS Microbiology Reviews published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  12. Yeast diversity and native vigor for flavor phenotypes.

    PubMed

    Carrau, Francisco; Gaggero, Carina; Aguilar, Pablo S

    2015-03-01

    Saccharomyces cerevisiae, the yeast used widely for beer, bread, cider, and wine production, is the most resourceful eukaryotic model used for genetic engineering. A typical concern about using engineered yeasts for food production might be negative consumer perception of genetically modified organisms. However, we believe the true pitfall of using genetically modified yeasts is their limited capacity to either refine or improve the sensory properties of fermented foods under real production conditions. Alternatively, yeast diversity screening to improve the aroma and flavors could offer groundbreaking opportunities in food biotechnology. We propose a 'Yeast Flavor Diversity Screening' strategy which integrates knowledge from sensory analysis and natural whole-genome evolution with information about flavor metabolic networks and their regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries.

    PubMed

    Curtin, Chris D; Bellon, Jennifer R; Henschke, Paul A; Godden, Peter W; de Barros Lopes, Miguel A

    2007-05-01

    Yeasts of the genus Dekkera and its anamorph Brettanomyces represent a significant spoilage issue for the global wine industry. Despite this, there is limited knowledge of genetic diversity and strain distribution within wine and winery-related environments. In this study, amplified fragment length polymorphism (AFLP) analysis was conducted on 244 Dekkera bruxellensis isolates from red wine made in 31 winemaking regions of Australia. The results indicated there were eight genotypes among the isolates, and three of these were commonly found across multiple winemaking regions. Analysis of 26S rRNA gene sequences provided further evidence of three common, conserved groups, whereas a phylogeny based upon the AFLP data demonstrated that the most common D. bruxellensis genotype (I) in Australian red wine was highly divergent from the D. bruxellensis type strain (CBS 74).

  14. Genetic diversity of the yeast Candida utilis.

    PubMed

    Stoltenburg, R; Klinner, U; Ritzerfeld, P; Zimmermann, M; Emeis, C C

    1992-12-01

    The electrophoretic karyotypes and some mtDNA restriction fragment patterns of 13 strains of Candida utilis and one strain of Hansenula jadinii were compared. PFGE separations revealed remarkable chromosome length polymorphisms between two groups of strains suggesting that perhaps they do not belong to the same species. However, all strains had the same or similar EcoRI, HindIII and BamHI mtDNA restriction patterns. The mtDNA genomes had an average size range of 55 kb. These results support the supposition that C. utilis is a yeast with a highly variable electrophoretic karyotype as already known for another imperfect yeast species, Candida albicans.

  15. Gene and genome construction in yeast.

    PubMed

    Gibson, Daniel G

    2011-04-01

    The yeast Saccharomyces cerevisiae has the capacity to take up and assemble dozens of different overlapping DNA molecules in one transformation event. These DNA molecules can be single-stranded oligonucleotides, to produce gene-sized fragments, or double-stranded DNA fragments, to produce molecules up to hundreds of kilobases in length, including complete bacterial genomes. This unit presents protocols for designing the DNA molecules to be assembled, transforming them into yeast, and confirming their assembly. © 2011 by John Wiley & Sons, Inc.

  16. Yeast diversity of sourdoughs and associated metabolic properties and functionalities.

    PubMed

    De Vuyst, Luc; Harth, Henning; Van Kerrebroeck, Simon; Leroy, Frédéric

    2016-12-19

    Together with acidifying lactic acid bacteria, yeasts play a key role in the production process of sourdough, where they are either naturally present or added as a starter culture. Worldwide, a diversity of yeast species is encountered, with Saccharomyces cerevisiae, Candida humilis, Kazachstania exigua, Pichia kudriavzevii, Wickerhamomyces anomalus, and Torulaspora delbrueckii among the most common ones. Sourdough-adapted yeasts are able to withstand the stress conditions encountered during their growth, including nutrient starvation as well as the effects of acidic, oxidative, thermal, and osmotic stresses. From a technological point of view, their metabolism primarily contributes to the leavening and flavour of sourdough products. Besides ethanol and carbon dioxide, yeasts can produce metabolites that specifically affect flavour, such as organic acids, diacetyl, higher alcohols from branched-chain amino acids, and esters derived thereof. Additionally, several yeast strains possess functional properties that can potentially lead to nutritional and safety advantages. These properties encompass the production of vitamins, an improvement of the bioavailability of phenolic compounds, the dephosphorylation of phytic acid, the presence of probiotic potential, and the inhibition of fungi and their mycotoxin production. Strains of diverse species are new candidate functional starter cultures, offering opportunities beyond the conventional use of baker's yeast.

  17. Yeast TATA-box transcription factor gene.

    PubMed

    Schmidt, M C; Kao, C C; Pei, R; Berk, A J

    1989-10-01

    The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.

  18. Studying Functions of All Yeast Genes Simultaneously

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Eason, Robert G.; Poumand, Nader; Herman, Zelek S.; Davis, Ronald W.; Anthony Kevin; Jejelowo, Olufisayo

    2006-01-01

    A method of studying the functions of all the genes of a given species of microorganism simultaneously has been developed in experiments on Saccharomyces cerevisiae (commonly known as baker's or brewer's yeast). It is already known that many yeast genes perform functions similar to those of corresponding human genes; therefore, by facilitating understanding of yeast genes, the method may ultimately also contribute to the knowledge needed to treat some diseases in humans. Because of the complexity of the method and the highly specialized nature of the underlying knowledge, it is possible to give only a brief and sketchy summary here. The method involves the use of unique synthetic deoxyribonucleic acid (DNA) sequences that are denoted as DNA bar codes because of their utility as molecular labels. The method also involves the disruption of gene functions through deletion of genes. Saccharomyces cerevisiae is a particularly powerful experimental system in that multiple deletion strains easily can be pooled for parallel growth assays. Individual deletion strains recently have been created for 5,918 open reading frames, representing nearly all of the estimated 6,000 genetic loci of Saccharomyces cerevisiae. Tagging of each deletion strain with one or two unique 20-nucleotide sequences enables identification of genes affected by specific growth conditions, without prior knowledge of gene functions. Hybridization of bar-code DNA to oligonucleotide arrays can be used to measure the growth rate of each strain over several cell-division generations. The growth rate thus measured serves as an index of the fitness of the strain.

  19. Genetic diversity and pectinolytic activity of epiphytic yeasts from grape carposphere.

    PubMed

    Filho, M Cilião; Bertéli, M B D; Valle, J S; Paccola-Meirelles, L D; Linde, G A; Barcellos, F G; Colauto, N B

    2017-06-20

    The genetic diversity of epiphytic yeasts from grape carposphere is susceptible to environmental variations that determine the predominant carposphere microbiota. Understanding the diversity of yeasts that inhabit grape carposphere in different environments and their pectinolytic activity is a way to understand the biotechnological potential that surrounds us and help improve winemaking. Therefore, this study aimed to evaluate the pectinolytic activity and characterize the genetic diversity of isolated epiphytic yeasts from grape carposphere. Grapes of the Bordeaux cultivar were collected from different regions of Paraná and Rio Grande do Sul States, in Brazil, and the yeasts were isolated from these grape carpospheres. Monosporic isolates were morphologically and genetically characterized on potato dextrose agar medium and by PCR-RFLP and rep-PCR (BOX-PCR) in the ITS1-5.8S-ITS2 region of rDNA. The index of pectinolytic activity of isolates was also evaluated estimating the ratio between the halo diameter of enzymatic degradation and the diameter of the colony when the isolates were grown in cultivation medium containing 10 g/L pectin, 5 g/L yeast extract, 15 g/L agar, 0.12% (w/v) Congo red, and pH 6.2. We observed that the grape carposphere is an environment with a great genetic diversity of epiphytic yeasts of the following genera: Cryptococcus (31.25%), Pichia (25.0%), Candida (25.0%), Dekkera (12.5%), and Saccharomyces (6.25%). The PCR-RFLP technique allowed analyzing existing polymorphism among individuals of a population based on a more restrict and evolutionarily preserved region, mostly utilized to differentiate isolates at the genus level. Approximately 33% of yeast isolates presented pectinolytic activity with potential biotechnological for wine and fruit juice production. This great genetic variability found indicated that it is a potential reservoir of genes to be applied in viniculture improvement programs.

  20. Yeast diversity on grapes in two German wine growing regions.

    PubMed

    Brysch-Herzberg, Michael; Seidel, Martin

    2015-12-02

    The yeast diversity on wine grapes in Germany, one of the most northern wine growing regions of the world, was investigated by means of a culture dependent approach. All yeast isolates were identified by sequence analysis of the D1/D2 domain of the 26S rDNA and the ITS region. Besides Hanseniaspora uvarum and Metschnikowia pulcherrima, which are well known to be abundant on grapes, Metschnikowia viticola, Rhodosporidium babjevae, and Curvibasidium pallidicorallinum, as well as two potentially new species related to Sporidiobolus pararoseus and Filobasidium floriforme, turned out to be typical members of the grape yeast community. We found M. viticola in about half of the grape samples in high abundance. Our data strongly suggest that M. viticola is one of the most important fermenting yeast species on grapes in the temperate climate of Germany. The frequent occurrence of Cu. pallidicorallinum and strains related to F. floriforme is a new finding. The current investigation provides information on the distribution of recently described yeast species, some of which are known from a very few strains up to now. Interestingly yeasts known for their role in the wine making process, such as Saccharomyces cerevisiae, Saccharomyces bayanus ssp. uvarum, Torulaspora delbrueckii, and Zygosaccharomyces bailii, were not found in the grape samples.

  1. Comparative genomics of wild type yeast strains unveils important genome diversity

    PubMed Central

    Carreto, Laura; Eiriz, Maria F; Gomes, Ana C; Pereira, Patrícia M; Schuller, Dorit; Santos, Manuel AS

    2008-01-01

    Background Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome

  2. Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts.

    PubMed

    Morel, Guillaume; Sterck, Lieven; Swennen, Dominique; Marcet-Houben, Marina; Onesime, Djamila; Levasseur, Anthony; Jacques, Noémie; Mallet, Sandrine; Couloux, Arnaux; Labadie, Karine; Amselem, Joëlle; Beckerich, Jean-Marie; Henrissat, Bernard; Van de Peer, Yves; Wincker, Patrick; Souciet, Jean-Luc; Gabaldón, Toni; Tinsley, Colin R; Casaregola, Serge

    2015-06-25

    The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi-yeasts split concomitant with the yeasts' genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

  3. Diversity of soil yeasts isolated from South Victoria Land, Antarctica

    USGS Publications Warehouse

    Connell, L.; Redman, R.; Craig, S.; Scorzetti, G.; Iszard, M.; Rodriguez, R.

    2008-01-01

    Unicellular fungi, commonly referred to as yeasts, were found to be components of the culturable soil fungal population in Taylor Valley, Mt. Discovery, Wright Valley, and two mountain peaks of South Victoria Land, Antarctica. Samples were taken from sites spanning a diversity of soil habitats that were not directly associated with vertebrate activity. A large proportion of yeasts isolated in this study were basidiomycetous species (89%), of which 43% may represent undescribed species, demonstrating that culturable yeasts remain incompletely described in these polar desert soils. Cryptococcus species represented the most often isolated genus (33%) followed by Leucosporidium (22%). Principle component analysis and multiple linear regression using stepwise selection was used to model the relation between abiotic variables (principle component 1 and principle component 2 scores) and yeast biodiversity (the number of species present at a given site). These analyses identified soil pH and electrical conductivity as significant predictors of yeast biodiversity. Species-specific PCR primers were designed to rapidly discriminate among the Dioszegia and Leucosporidium species collected in this study. ?? 2008 Springer Science+Business Media, LLC.

  4. Optogenetic switches for light-controlled gene expression in yeast.

    PubMed

    Salinas, Francisco; Rojas, Vicente; Delgado, Verónica; Agosin, Eduardo; Larrondo, Luis F

    2017-04-01

    Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

  5. Complementation of Yeast Genes with Human Genes as an Experimental Platform for Functional Testing of Human Genetic Variants.

    PubMed

    Hamza, Akil; Tammpere, Erik; Kofoed, Megan; Keong, Christelle; Chiang, Jennifer; Giaever, Guri; Nislow, Corey; Hieter, Philip

    2015-11-01

    While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human-yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach. Copyright © 2015 by the Genetics Society of America.

  6. Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

    PubMed Central

    Mills, David A.; Johannsen, Eric A.; Cocolin, Luca

    2002-01-01

    Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (∼30°C) and ambient (∼20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>106 cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations. PMID:12324335

  7. The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations.

    PubMed

    Fernández Maura, Yurelkys; Balzarini, Tom; Clapé Borges, Pablo; Evrard, Pierre; De Vuyst, Luc; Daniel, H-M

    2016-09-16

    The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have

  8. The sensitive [SWI (+)] prion: new perspectives on yeast prion diversity.

    PubMed

    Hines, Justin K; Craig, Elizabeth A

    2011-01-01

    Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these "nouveau prions" has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI(+)], differs from the best studied, archetypal prion [PSI(+)] in several significant ways. ( 1) Notably, [SWI(+)] is highly sensitive to alterations in Hsp70 system chaperone activity and is lost upon growth at elevated temperatures. In that report we briefly noted a correlation amongst prions regarding amino acid composition, seed number and sensitivity to the activity of the Hsp70 chaperone system. Here we extend that analysis and put forth the idea that [SWI(+)] may be representative of a class of asparagine-rich yeast prions which also includes [URE3], [MOT3(+)] and [ISP(+)], distinct from the glutamine-rich prions such as [PSI(+)] and [RNQ(+)]. While much work remains, it is apparent that our understanding of the extent of the diversity of prion characteristics is in its infancy.

  9. Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts

    PubMed Central

    Morel, Guillaume; Sterck, Lieven; Swennen, Dominique; Marcet-Houben, Marina; Onesime, Djamila; Levasseur, Anthony; Jacques, Noémie; Mallet, Sandrine; Couloux, Arnaux; Labadie, Karine; Amselem, Joëlle; Beckerich, Jean-Marie; Henrissat, Bernard; Van de Peer, Yves; Wincker, Patrick; Souciet, Jean-Luc; Gabaldón, Toni; Tinsley, Colin R.; Casaregola, Serge

    2015-01-01

    The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi–yeasts split concomitant with the yeasts’ genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts. PMID:26108467

  10. Yeast diversity of Ghanaian cocoa bean heap fermentations.

    PubMed

    Daniel, Heide-Marie; Vrancken, Gino; Takrama, Jemmy F; Camu, Nicholas; De Vos, Paul; De Vuyst, Luc

    2009-08-01

    The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii (Issatchenkia orientalis), Saccharomyces cerevisiae, and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma (Pichia) caribbica, Pichia manshurica, Saccharomycodes ludwigii, and Yamadazyma (Pichia) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae.

  11. In vivo continuous evolution of genes and pathways in yeast

    PubMed Central

    Crook, Nathan; Abatemarco, Joseph; Sun, Jie; Wagner, James M.; Schmitz, Alexander; Alper, Hal S.

    2016-01-01

    Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107 l−1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo. PMID:27748457

  12. French Jura flor yeasts: genotype and technological diversity.

    PubMed

    Charpentier, Claudine; Colin, Anne; Alais, Anne; Legras, Jean-Luc

    2009-03-01

    Fifty-four Saccharomyces cerevisiae strains were isolated from Jura "Vin Jaune" velum and characterized by conventional physiological and molecular tests including ITS RFLP and sequence analysis, karyotyping and inter delta typing. ITS RFLP and sequence revealed a specific group of related strains different from the specific profile of Sherry flor yeast caused by a 24 bp deletion in the ITS1 region described by Esteve-Zarzoso et al. (Antonie Van Leeuwenhoek 85:151-158, 2004). Interdelta typing, the most discriminative method, revealed a high diversity of Jura flor yeast strains and gathered strains in clusters unequally shared between the northern and southern part of the Jura vineyard. The assessment of phenotypic diversity among the isolated strains was investigated for three wine metabolites (ethanal, acetic acid, and sotolon) from micro scale velum tests. Except at an early stage of ageing, the production of these metabolites was not correlated to the five genetic groups obtained by interdelta typing, but correlated to the cellar where strains had been isolated. The different strains isolated in a cellar produced mostly one type of velum (thin or thick, grey or white); but thin and grey velums, recognized as responsible for high quality wines, were obtained more frequently for one of the five groups of delta genotypes.

  13. Fine structure of Tibetan kefir grains and their yeast distribution, diversity, and shift.

    PubMed

    Lu, Man; Wang, Xingxing; Sun, Guowei; Qin, Bing; Xiao, Jinzhou; Yan, Shuling; Pan, Yingjie; Wang, Yongjie

    2014-01-01

    Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species--Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S

  14. Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift

    PubMed Central

    Lu, Man; Wang, Xingxing; Sun, Guowei; Qin, Bing; Xiao, Jinzhou; Yan, Shuling; Pan, Yingjie; Wang, Yongjie

    2014-01-01

    Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S

  15. Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal.

    PubMed

    Gadanho, Mário; Sampaio, José Paulo

    2004-12-01

    Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.

  16. Posttranscriptional Control of Gene Expression in Yeast

    PubMed Central

    McCarthy, John E. G.

    1998-01-01

    Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our understanding of the posttranscriptional steps of eukaryotic gene expression. Given the wide range of experimental tools applicable to S. cerevisiae and the recent determination of its complete genomic sequence, many of the key challenges of the posttranscriptional control field can be tackled particularly effectively by using this organism. This article reviews the current knowledge of the cellular components and mechanisms related to translation and mRNA decay, with the emphasis on the molecular basis for rate control and gene regulation. Recent progress in characterizing translation factors and their protein-protein and RNA-protein interactions has been rapid. Against the background of a growing body of structural information, the review discusses the thermodynamic and kinetic principles that govern the translation process. As in prokaryotic systems, translational initiation is a key point of control. Modulation of the activities of translational initiation factors imposes global regulation in the cell, while structural features of particular 5′ untranslated regions, such as upstream open reading frames and effector binding sites, allow for gene-specific regulation. Recent data have revealed many new details of the molecular mechanisms involved while providing insight into the functional overlaps and molecular networking that are apparently a key feature of evolving cellular systems. An overall picture of the mechanisms governing mRNA decay has only very recently begun to develop. The latest work has revealed new information about the mRNA decay pathways, the components of the mRNA degradation machinery, and the way in which these might relate to the translation apparatus. Overall, major challenges still to be addressed include the task of relating principles of posttranscriptional control to cellular compartmentalization and polysome structure and the role of molecular channelling

  17. Transferring a synthetic gene circuit from yeast to mammalian cells.

    PubMed

    Nevozhay, Dmitry; Zal, Tomasz; Balázsi, Gábor

    2013-01-01

    The emerging field of synthetic biology builds gene circuits for scientific, industrial and therapeutic needs. Adaptability of synthetic gene circuits across different organisms could enable a synthetic biology pipeline, where circuits are designed in silico, characterized in microbes and reimplemented in mammalian settings for practical usage. However, the processes affecting gene circuit adaptability have not been systematically investigated. Here we construct a mammalian version of a negative feedback-based 'linearizer' gene circuit previously developed in yeast. The first naïve mammalian prototype was non-functional, but a computational model suggested that we could recover function by improving gene expression and protein localization. After rationally developing and combining new parts as the model suggested, we regained function and could tune target gene expression in human cells linearly and precisely as in yeast. The steps we have taken should be generally relevant for transferring any gene circuit from yeast into mammalian cells.

  18. The yeast ubiquitin genes: a family of natural gene fusions.

    PubMed Central

    Ozkaynak, E; Finley, D; Solomon, M J; Varshavsky, A

    1987-01-01

    Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress. Images Fig. 1. Fig. 7. PMID:3038523

  19. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Chen, C Y; Oppermann, H; Hitzeman, R A

    1984-01-01

    DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels. Images PMID:6096814

  20. Presence of STA gene sequences in brewer's yeast genome.

    PubMed

    Balogh, I; Maráz, A

    1996-06-01

    STA genes are responsible for producing extracellular glucoamylase enzymes in Saccharomyces cerevisiae var. diastaticus. These genes exist in three forms, which are located on three different chromosomes. The nucleotide sequences of the STA genes are highly homologous. A sporulation-specific glucoamylase gene called SGA1 exists in every Saccharomyces cerevisiae strain, this also having a partly homologous DNA sequence with the STA genes. In this study S. cerevisiae var. diastaticus and brewer's yeast strains were characterized by pulsed-field gel electrophoresis. In many cases chromosome length polymorphism (CLP) was found. The chromosomes were hybridized with a DNA probe which was homologous with STA genes and the SGA1 gene. Presence of the SGA1 gene was detected in each strain used. Four brewing yeasts were found to have homologous sequences with the STA3 gene on chromosome XIV despite the fact that these strains were not able to produce extracellular glucoamylase enzyme.

  1. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing

    PubMed Central

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A.

    2014-01-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. PMID:24814796

  2. Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

    PubMed

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A; Verstrepen, Kevin J; Lievens, Bart

    2014-07-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. A Gondwanan imprint on global diversity and domestication of wine and cider yeast Saccharomyces uvarum.

    PubMed

    Almeida, Pedro; Gonçalves, Carla; Teixeira, Sara; Libkind, Diego; Bontrager, Martin; Masneuf-Pomarède, Isabelle; Albertin, Warren; Durrens, Pascal; Sherman, David James; Marullo, Philippe; Hittinger, Chris Todd; Gonçalves, Paula; Sampaio, José Paulo

    2014-06-02

    In addition to Saccharomyces cerevisiae, the cryotolerant yeast species S. uvarum is also used for wine and cider fermentation but nothing is known about its natural history. Here we use a population genomics approach to investigate its global phylogeography and domestication fingerprints using a collection of isolates obtained from fermented beverages and from natural environments on five continents. South American isolates contain more genetic diversity than that found in the Northern Hemisphere. Moreover, coalescence analyses suggest that a Patagonian sub-population gave rise to the Holarctic population through a recent bottleneck. Holarctic strains display multiple introgressions from other Saccharomyces species, those from S. eubayanus being prevalent in European strains associated with human-driven fermentations. These introgressions are absent in the large majority of wild strains and gene ontology analyses indicate that several gene categories relevant for wine fermentation are overrepresented. Such findings constitute a first indication of domestication in S. uvarum.

  4. A Gondwanan Imprint on Global Diversity and Domestication of Wine and Cider Yeast Saccharomyces uvarum

    PubMed Central

    Almeida, Pedro; Gonçalves, Carla; Teixeira, Sara; Libkind, Diego; Bontrager, Martin; Masneuf-Pomarède, Isabelle; Albertin, Warren; Durrens, Pascal; Sherman, David; Marullo, Philippe; Hittinger, Chris Todd; Gonçalves, Paula; Sampaio, José Paulo

    2016-01-01

    In addition to Saccharomyces cerevisiae, the cryotolerant yeast species S. uvarum is also used for wine and cider fermentation but nothing is known about its natural history. Here we use a population genomics approach to investigate its global phylogeography and domestication fingerprints using a collection of isolates obtained from fermented beverages and from natural environments on five continents. South American isolates contain more genetic diversity than that found in the Northern Hemisphere. Moreover, coalescence analyses suggest that a Patagonian sub-population gave rise to the Holarctic population through a recent bottleneck. Holarctic strains display multiple introgressions from other Saccharomyces species, those from S. eubayanus being prevalent in European strains associated with human-driven fermentations. These introgressions are absent in the large majority of wild strains and gene ontology analyses indicate that several gene categories relevant for wine fermentation are overrepresented. Such findings constitute a first indication of domestication in S. uvarum. PMID:24887054

  5. A Gondwanan imprint on global diversity and domestication of wine and cider yeast Saccharomyces uvarum

    NASA Astrophysics Data System (ADS)

    Almeida, Pedro; Gonçalves, Carla; Teixeira, Sara; Libkind, Diego; Bontrager, Martin; Masneuf-Pomarède, Isabelle; Albertin, Warren; Durrens, Pascal; Sherman, David James; Marullo, Philippe; Todd Hittinger, Chris; Gonçalves, Paula; Sampaio, José Paulo

    2014-06-01

    In addition to Saccharomyces cerevisiae, the cryotolerant yeast species S. uvarum is also used for wine and cider fermentation but nothing is known about its natural history. Here we use a population genomics approach to investigate its global phylogeography and domestication fingerprints using a collection of isolates obtained from fermented beverages and from natural environments on five continents. South American isolates contain more genetic diversity than that found in the Northern Hemisphere. Moreover, coalescence analyses suggest that a Patagonian sub-population gave rise to the Holarctic population through a recent bottleneck. Holarctic strains display multiple introgressions from other Saccharomyces species, those from S. eubayanus being prevalent in European strains associated with human-driven fermentations. These introgressions are absent in the large majority of wild strains and gene ontology analyses indicate that several gene categories relevant for wine fermentation are overrepresented. Such findings constitute a first indication of domestication in S. uvarum.

  6. NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts.

    PubMed Central

    Nosek, J; Fukuhara, H

    1994-01-01

    The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts. In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6. The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe. Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria. Hybridization experiments using C. parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others. Images PMID:7521869

  7. Enlightenment of Yeast Mitochondrial Homoplasmy: Diversified Roles of Gene Conversion

    PubMed Central

    Ling, Feng; Mikawa, Tsutomu; Shibata, Takehiko

    2011-01-01

    Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes. PMID:24710143

  8. Yeast diversity associated to sediments and water from two Colombian artificial lakes.

    PubMed

    Silva-Bedoya, L M; Ramírez-Castrillón, M; Osorio-Cadavid, E

    2014-01-01

    In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them.

  9. Yeast diversity associated to sediments and water from two Colombian artificial lakes

    PubMed Central

    Silva-Bedoya, L.M.; Ramírez-Castrillón, M.; Osorio-Cadavid, E.

    2014-01-01

    In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them. PMID:24948924

  10. The influence of Aster x salignus Willd. Invasion on the diversity of soil yeast communities

    NASA Astrophysics Data System (ADS)

    Glushakova, A. M.; Kachalkin, A. V.; Chernov, I. Yu.

    2016-07-01

    The annual dynamics of yeast communities were studied in the soddy-podzolic soil under the thickets of Aster x salignus Willd., one of the widespread invasive plant species in central Russia. Yeast groups in the soils under continuous aster thickets were found to differ greatly from the yeast communities in the soils under the adjacent indigenous meadow vegetation. In both biotopes the same species ( Candida vartiovaarae, Candida sake, and Cryptococcus terreus) are dominants. However, in the soils under indigenous grasses, eurybiontic yeasts Rhodotorula mucilaginosa, which almost never occur in the soil under aster, are widespread. In the soil under aster, the shares of other typical epiphytic and pedobiontic yeast fungi (ascomycetic species Wickerhamomyces aniomalus, Barnettozyma californica and basidiomycetic species Cystofilobasidium macerans, Guehomyces pullulans) significantly increase. Thus, the invasion of Aster x salignus has a clear effect on soil yeast complexes reducing their taxonomic and ecological diversity.

  11. A yeast-based rapid prototype platform for gene control elements in mammalian cells.

    PubMed

    Wei, Kathy Y; Chen, Yvonne Y; Smolke, Christina D

    2013-04-01

    Programming genetic circuits in mammalian cells requires flexible, tunable, and user-tailored gene-control systems. However, most existing control systems are either mechanistically specific for microbial organisms or must be laboriously re-engineered to function in mammalian cells. Here, we demonstrate a ribozyme-based device platform that can be directly transported from yeast to mammalian cells in a "plug-and-play" manner. Ribozyme switches previously prototyped in yeast are shown to regulate gene expression in a predictable, ligand-responsive manner in human HEK 293, HeLa, and U2OS cell lines without any change to device sequence nor further optimization. The ribozyme-based devices, which exhibit activation ratios comparable to the best RNA-based regulatory devices demonstrated in mammalian cells to-date, retain their prescribed functions (ON or OFF switch), tunability of regulatory stringency, and responsiveness to different small-molecule inputs in mammalian hosts. Furthermore, we observe strong correlations of device performance between yeast and all mammalian cell lines tested (R(2)  = 0.63-0.97). Our unique device architecture can therefore act as a rapid prototyping platform (RPP) based on a yeast chassis, providing a well-developed and genetically tractable system that supports rapid and high-throughput screens for generating gene-controllers with a broad range of functions in mammalian cells. This platform will accelerate development of mammalian gene-controllers for diverse applications, including cell-based therapeutics and cell-fate reprogramming.

  12. A Complete Set of Nascent Transcription Rates for Yeast Genes

    PubMed Central

    Pelechano, Vicent; Chávez, Sebastián; Pérez-Ortín, José E.

    2010-01-01

    The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) and the RNA pol ChIP-on-chip methods we generated a new, more accurate nascent transcription rate dataset. By comparing this dataset with the indirect ones obtained from the mRNA stabilities and mRNA amount datasets, we are able to obtain biological information about posttranscriptional regulation processes and a genomic snapshot of the location of the active transcriptional machinery. We have obtained nascent transcription rates for 4,670 yeast genes. The median RNA polymerase II density in the genes is 0.078 molecules/kb, which corresponds to an average of 0.096 molecules/gene. Most genes have transcription rates of between 2 and 30 mRNAs/hour and less than 1% of yeast genes have >1 RNA polymerase molecule/gene. Histone and ribosomal protein genes are the highest transcribed groups of genes and other than these exceptions the transcription of genes is an infrequent phenomenon in a yeast cell. PMID:21103382

  13. Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

    PubMed

    Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

    2017-10-01

    Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Advances in Gene Expression in Non-Conventional Yeasts

    NASA Astrophysics Data System (ADS)

    Nel, Sanet; Labuschagne, Michel; Albertyn, Jacobus

    Yeast has been a favoured lower eukaryotic system for the expression and production of recombinant proteins for both basic research and practical applications, and the demand for foreign-gene expression systems is increasing rapidly. Despite the vast amount of information on the molecular biology and physiology of Saccharomyces cerevisiae, which has consequently been the first choice as host system for recombinant protein production in the past, several limitations have been identified in this expression system. These limitations have recently been relieved by the development of expression systems in other yeast species known as ‘ non-conventional yeasts’ or ‘non-Saccharomyces ’ yeasts. With the increasing interest in the biotechnological applications of these yeasts in applied and fundamental studies and processes, the term ‘ non-conventional ’ yeast may well soon become redundant. As there is no universal expression system for heterologous protein production, it is necessary to recognize the merits and demerits of each system in order to make a right choice. This chapter will evaluate the competitive environment of non-conventional expression platforms represented by some of the best-known alternative yeasts systems including Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris and more recently, Arxula adeninivorans.

  15. Seasonal and altitudinal changes of culturable bacterial and yeast diversity in Alpine forest soils.

    PubMed

    França, Luís; Sannino, Ciro; Turchetti, Benedetta; Buzzini, Pietro; Margesin, Rosa

    2016-11-01

    The effect of altitude and season on abundance and diversity of the culturable heterotrophic bacterial and yeast community was examined at four forest sites in the Italian Alps along an altitude gradient (545-2000 m). Independently of altitude, bacteria isolated at 0 °C (psychrophiles) were less numerous than those recovered at 20 °C. In autumn, psychrophilic bacterial population increased with altitude. The 1194 bacterial strains were primarily affiliated with the classes Alpha-, Beta-, Gammaproteobacteria, Spingobacteriia and Flavobacteriia. Fifty-seven of 112 operational taxonomic units represented potential novel species. Strains isolated at 20 °C had a higher diversity and showed similarities in taxa composition and abundance, regardless of altitude or season, while strains isolated at 0 °C showed differences in community composition at lower and higher altitudes. In contrast to bacteria, yeast diversity was season-dependent: site- and altitude-specific effects on yeast diversity were only detected in spring. Isolation temperature affected the relative proportions of yeast genera. Isolations recovered 719 strains, belonging to the classes Dothideomycetes, Saccharomycetes, Tremellomycetes and Mycrobotryomycetes. The presence of few dominant bacterial OTUs and yeast species indicated a resilient microbial population that is not affected by season or altitude. Soil nutrient contents influenced significantly abundance and diversity of culturable bacteria, but not of culturable yeasts.

  16. Occurrence and diversity of marine yeasts in Antarctica environments

    NASA Astrophysics Data System (ADS)

    Zhang, Xue; Hua, Mingxia; Song, Chunli; Chi, Zhenming

    2012-03-01

    A total of 28 yeast strains were obtained from the sea sediment of Antarctica. According to the results of routine identification and molecular characterization, the strains belonged to species of Yarrowia lipolytica, Debaryomyces hansenii, Rhodotorula slooffiae, Rhodotorula mucilaginosa, Sporidiobolus salmonicolor, Aureobasidium pullulans, Mrakia frigida and Guehomyces pullulans, respectively. The Antarctica yeasts have wide potential applications in biotechnology, for some of them can produce β-galactosidase and killer toxins.

  17. Global analysis of fission yeast mating genes reveals new autophagy factors.

    PubMed

    Sun, Ling-Ling; Li, Ming; Suo, Fang; Liu, Xiao-Man; Shen, En-Zhi; Yang, Bing; Dong, Meng-Qiu; He, Wan-Zhong; Du, Li-Lin

    2013-01-01

    Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12-Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.

  18. Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

    PubMed Central

    Sun, Ling-Ling; Shen, En-Zhi; Yang, Bing; Dong, Meng-Qiu; He, Wan-Zhong; Du, Li-Lin

    2013-01-01

    Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy. PMID:23950735

  19. Identification of cell cycle-regulated genes in fission yeast.

    PubMed

    Peng, Xu; Karuturi, R Krishna Murthy; Miller, Lance D; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T; Balasubramanian, Mohan K; Liu, Jianhua

    2005-03-01

    Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found approximately 140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.

  20. Yeast metabolic chassis designs for diverse biotechnological products

    PubMed Central

    Jouhten, Paula; Boruta, Tomasz; Andrejev, Sergej; Pereira, Filipa; Rocha, Isabel; Patil, Kiran Raosaheb

    2016-01-01

    The diversity of industrially important molecules for which microbial production routes have been experimentally demonstrated is rapidly increasing. The development of economically viable producer cells is, however, lagging behind, as it requires substantial engineering of the host metabolism. A chassis strain suitable for production of a range of molecules is therefore highly sought after but remains elusive. Here, we propose a genome-scale metabolic modeling approach to design chassis strains of Saccharomyces cerevisiae – a widely used microbial cell factory. For a group of 29 products covering a broad range of biochemistry and applications, we identified modular metabolic engineering strategies for re-routing carbon flux towards the desired product. We find distinct product families with shared targets forming the basis for the corresponding chassis cells. The design strategies include overexpression targets that group products by similarity in precursor and cofactor requirements, as well as gene deletion strategies for growth-product coupling that lead to non-intuitive product groups. Our results reveal the extent and the nature of flux re-routing necessary for producing a diverse range of products in a widely used cell factory and provide blueprints for constructing pre-optimized chassis strains. PMID:27430744

  1. Yeast metabolic chassis designs for diverse biotechnological products.

    PubMed

    Jouhten, Paula; Boruta, Tomasz; Andrejev, Sergej; Pereira, Filipa; Rocha, Isabel; Patil, Kiran Raosaheb

    2016-07-19

    The diversity of industrially important molecules for which microbial production routes have been experimentally demonstrated is rapidly increasing. The development of economically viable producer cells is, however, lagging behind, as it requires substantial engineering of the host metabolism. A chassis strain suitable for production of a range of molecules is therefore highly sought after but remains elusive. Here, we propose a genome-scale metabolic modeling approach to design chassis strains of Saccharomyces cerevisiae - a widely used microbial cell factory. For a group of 29 products covering a broad range of biochemistry and applications, we identified modular metabolic engineering strategies for re-routing carbon flux towards the desired product. We find distinct product families with shared targets forming the basis for the corresponding chassis cells. The design strategies include overexpression targets that group products by similarity in precursor and cofactor requirements, as well as gene deletion strategies for growth-product coupling that lead to non-intuitive product groups. Our results reveal the extent and the nature of flux re-routing necessary for producing a diverse range of products in a widely used cell factory and provide blueprints for constructing pre-optimized chassis strains.

  2. Natural gene expression variation studies in yeast.

    PubMed

    Thompson, Dawn A; Cubillos, Francisco A

    2017-01-01

    The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants, mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap, such as mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, probably owing to genetic divergence. Future studies in this area will benefit from the access to a massive array of well-annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Identifying genes required for respiratory growth of fission yeast

    PubMed Central

    2016-01-01

    We have used both auxotroph and prototroph versions of the latest deletion-mutant library to identify genes required for respiratory growth on solid glycerol medium in fission yeast. This data set complements and enhances our recent study on functional and regulatory aspects of energy metabolism by providing additional proteins that are involved in respiration. Most proteins identified in this mutant screen have not been implicated in respiration in budding yeast. We also provide a protocol to generate a prototrophic mutant library, and data on technical and biological reproducibility of colony-based high-throughput screens. PMID:27918601

  4. Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast

    PubMed Central

    Contamine, Véronique; Picard, Marguerite

    2000-01-01

    Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho+ mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho0 petites) and/or lead to truncated forms (rho−) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho+ genome depends on a centromere-like structure dispensable for the maintenance of rho− mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes. PMID:10839818

  5. Oral yeast flora and its ITS sequence diversity among a large cohort of medical students in Hainan, China.

    PubMed

    Wang, Huamin; Wang, Yin; Chen, Jinglong; Zhan, Zilong; Li, Yinglin; Xu, Jianping

    2007-08-01

    The most prevalent fungal infection of humans is candidiasis which is caused by species of Candida that are typical members of the commensal microbial flora of the oral mucosa and other body surfaces. Since species of Candida differ in virulence properties and susceptibilities to anti-fungal drugs, understanding the human commensal yeast flora will have a significant impact on designing treatment and prevention strategies against yeast infections. However, although there is a global interest in Candida species, the global distributions of Candida species remain largely unknown, especially among healthy hosts. Here we report the oral yeast flora from the surveys of over 1,000 medical students in China. Our results showed that this population had a yeast carriage rate (4.5%) much lower than other population samples reported previously from Mainland China (40-70%). In addition, C. albicans was isolated at a much higher frequency than those from other Chinese samples, with a frequency (80.9%) more similar to those in developed regions such as North America. The oral yeast carriage rates and yeast species compositions were similar between male and female students and between the hosts borne and raised on Hainan Island and those borne and raised on Mainland China. Furthermore, the sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster was analyzed for strains of the dominant species, C. albicans. Our analysis identified 14 ITS types among the 41 Hainan isolates of C. albicans. However, only four of the 14 ITS types were identical to those in reference strains from Europe and North America. Taken together, our analyses suggest that the oral yeast flora among host populations in China is highly heterogeneous and that there is a high ITS sequence diversity in the Hainan population of C. albicans.

  6. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Kim, Dong-Uk; Hayles, Jacqueline; Kim, Dongsup; Wood, Valerie; Park, Han-Oh; Won, Misun; Yoo, Hyang-Sook; Duhig, Trevor; Nam, Miyoung; Palmer, Georgia; Han, Sangjo; Jeffery, Linda; Baek, Seung-Tae; Lee, Hyemi; Shim, Young Sam; Lee, Minho; Kim, Lila; Heo, Kyung-Sun; Noh, Eun Joo; Lee, Ah-Reum; Jang, Young-Joo; Chung, Kyung-Sook; Choi, Shin-Jung; Park, Jo-Young; Park, Youngwoo; Kim, Hwan Mook; Park, Song-Kyu; Park, Hae-Joon; Kang, Eun-Jung; Kim, Hyong Bai; Kang, Hyun-Sam; Park, Hee-Moon; Kim, Kyunghoon; Song, Kiwon; Song, Kyung Bin; Nurse, Paul; Hoe, Kwang-Lae

    2010-06-01

    We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

  7. Yeast diversity in the acidic Rio Agrio-Lake Caviahue volcanic environment (Patagonia, Argentina).

    PubMed

    Russo, Gabriel; Libkind, Diego; Sampaio, José P; van Broock, Maria R

    2008-09-01

    The Rio Agrio and Lake Caviahue system (RAC), in Northwestern Patagonia, is a natural acidic environment. The aims of this study were to characterize the yeast community and to provide the first ecological assessment of yeast diversity of this extreme aquatic environment. Yeast occurrence and diversity were studied at seven sites where the water pH varied between 1.8 and 6.7. Yeast CFU counts in the river ranged from 30 to 1200 CFU L(-1), but in the Lake the values were lower (30-60 CFU L(-1)). A total of 25 different yeast species were found, 11 of which belonged to undescribed taxa. Among these was an unusual strongly acidophilic Cryptococcus species. The RAC yeast community resembles that of acidic aquatic environments resulting from anthropic activities such as the São Domingos mines in Portugal and the Rio Tinto in Spain, respectively. The isolated yeast species were organized into different grades of adaptation to the RAC aquatic system. Based on the proposed grades, Rhodotorula mucilaginosa, Rhodosporidium toruloides and two novel Cryptococcus species were the most adapted species. These Cryptococcus species are apparently specialists of acidic aquatic environments, and might bear physiological features that possibly account for their ability to thrive in such extreme environments.

  8. Local climatic conditions constrain soil yeast diversity patterns in Mediterranean forests, woodlands and scrub biome.

    PubMed

    Yurkov, Andrey M; Röhl, Oliver; Pontes, Ana; Carvalho, Cláudia; Maldonado, Cristina; Sampaio, José Paulo

    2016-02-01

    Soil yeasts represent a poorly known fraction of the soil microbiome due to limited ecological surveys. Here, we provide the first comprehensive inventory of cultivable soil yeasts in a Mediterranean ecosystem, which is the leading biodiversity hotspot for vascular plants and vertebrates in Europe. We isolated and identified soil yeasts from forested sites of Serra da Arrábida Natural Park (Portugal), representing the Mediterranean forests, woodlands and scrub biome. Both cultivation experiments and the subsequent species richness estimations suggest the highest species richness values reported to date, resulting in a total of 57 and 80 yeast taxa, respectively. These values far exceed those reported for other forest soils in Europe. Furthermore, we assessed the response of yeast diversity to microclimatic environmental factors in biotopes composed of the same plant species but showing a gradual change from humid broadleaf forests to dry maquis. We observed that forest properties constrained by precipitation level had strong impact on yeast diversity and on community structure and lower precipitation resulted in an increased number of rare species and decreased evenness values. In conclusion, the structure of soil yeast communities mirrors the environmental factors that affect aboveground phytocenoses, aboveground biomass and plant projective cover.

  9. The diversity of yeasts associated with grapes and musts of the Strekov winegrowing region, Slovakia.

    PubMed

    Nemcová, Kornélia; Breierová, Emília; Vadkertiová, Renáta; Molnárová, Jana

    2015-03-01

    Many different yeast species have been isolated from grapes and musts worldwide. The diversity and frequency of yeasts depend on a number of factors such as the grape variety, the physical damage of the grapes, the weather conditions and the chemical composition of must. A total of 366 isolates were associated with the three grape cultivars: Blue Frankish, Green Veltliner and Sauvignon blanc over four consecutive years. Yeast cultures were isolated from the grapes and from the fermenting musts after the first and seventh days. The ascomycetous yeasts of the genera Aureobasidium, Candida, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Saccharomycopsis together with basidiomycetous yeasts of the genera Cryptococcus, Dioszegia, Filobasidium, Rhodotorula and Sporidiobolus were associated with the three grape varieties. Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia kluyveri, Pichia kudriavzevii and Sporidiobolus pararoseus were found on the berries in significant amounts. P. kluyveri and P. kudriavzevii were more associated with the damaged grapes, whereas Sp. pararoseus with intact ones. H. uvarum and M. pulcherrima were present on both types of grapes almost equally. The yeast composition and quantitative representation of yeast species varied over the grape varieties and the years examined. Although the basidiomycetous species formed a significant proportion of the yeast population in some individual grape variety/year combinations, the ascomycetous species were dominant.

  10. Yeast Communities of Diverse Drosophila Species: Comparison of Two Symbiont Groups in the Same Hosts

    PubMed Central

    Eisen, Jonathan A.; Kopp, Artyom

    2012-01-01

    The combination of ecological diversity with genetic and experimental tractability makes Drosophila a powerful model for the study of animal-associated microbial communities. Despite the known importance of yeasts in Drosophila physiology, behavior, and fitness, most recent work has focused on Drosophila-bacterial interactions. In order to get a more complete understanding of the Drosophila microbiome, we characterized the yeast communities associated with different Drosophila species collected around the world. We focused on the phylum Ascomycota because it constitutes the vast majority of the Drosophila-associated yeasts. Our sampling strategy allowed us to compare the distribution and structure of the yeast and bacterial communities in the same host populations. We show that yeast communities are dominated by a small number of abundant taxa, that the same yeast lineages are associated with different host species and populations, and that host diet has a greater effect than host species on yeast community composition. These patterns closely parallel those observed in Drosophila bacterial communities. However, we do not detect a significant correlation between the yeast and bacterial communities of the same host populations. Comparative analysis of different symbiont groups provides a more comprehensive picture of host-microbe interactions. Future work on the role of symbiont communities in animal physiology, ecological adaptation, and evolution would benefit from a similarly holistic approach. PMID:22885750

  11. Periodic gene expression program of the fission yeast cell cycle.

    PubMed

    Rustici, Gabriella; Mata, Juan; Kivinen, Katja; Lió, Pietro; Penkett, Christopher J; Burns, Gavin; Hayles, Jacqueline; Brazma, Alvis; Nurse, Paul; Bähler, Jürg

    2004-08-01

    Cell-cycle control of transcription seems to be universal, but little is known about its global conservation and biological significance. We report on the genome-wide transcriptional program of the Schizosaccharomyces pombe cell cycle, identifying 407 periodically expressed genes of which 136 show high-amplitude changes. These genes cluster in four major waves of expression. The forkhead protein Sep1p regulates mitotic genes in the first cluster, including Ace2p, which activates transcription in the second cluster during the M-G1 transition and cytokinesis. Other genes in the second cluster, which are required for G1-S progression, are regulated by the MBF complex independently of Sep1p and Ace2p. The third cluster coincides with S phase and a fourth cluster contains genes weakly regulated during G2 phase. Despite conserved cell-cycle transcription factors, differences in regulatory circuits between fission and budding yeasts are evident, revealing evolutionary plasticity of transcriptional control. Periodic transcription of most genes is not conserved between the two yeasts, except for a core set of approximately 40 genes that seem to be universally regulated during the eukaryotic cell cycle and may have key roles in cell-cycle progression.

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

    NASA Technical Reports Server (NTRS)

    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.

  13. Yeast diversity in the extreme acidic environments of the Iberian Pyrite Belt.

    PubMed

    Gadanho, Mário; Libkind, Diego; Sampaio, José Paulo

    2006-10-01

    In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.

  14. Diversity of yeast species during fermentative process contributing to Chinese Maotai-flavour liquor making.

    PubMed

    Wu, Q; Xu, Y; Chen, L

    2012-10-01

    The famous traditional Chinese Maotai-flavour liquor is produced by a unique spontaneous simultaneous saccharification and fermentation process, which contributes to a distinctive yeast community with specific physiological properties and performances. Therefore, it would be useful to investigate this yeast community and reveal the novelty of its characteristics. Nine yeast species were obtained from the fermentation period. A combination of physiological and functional analyses revealed a very high diversity of yeast populations. In particular, the extremely high temperature and low acidity of the fermentation conditions led to an accumulation of species with distinctive heat- and acid-resistant properties. Moreover, these yeast species were also significant flavour contributors, for various alcohols, acids and esters. The Chinese Maotai-flavour liquor-fermentation process is rich in yeast resources with distinctive fermentation properties, which were different from other beverages. This work is the first to study the complex yeast ecology of the Maotai-flavour liquor-making process. It allows a deeper insight into the mechanism of this process. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  15. Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines.

    PubMed

    Bellon, Jennifer R; Eglinton, Jeffery M; Siebert, Tracey E; Pollnitz, Alan P; Rose, Louisa; de Barros Lopes, Miguel; Chambers, Paul J

    2011-08-01

    Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.

  16. A hammerhead ribozyme inhibits ADE1 gene expression in yeast.

    PubMed

    Ferbeyre, G; Bratty, J; Chen, H; Cedergren, R

    1995-03-21

    To study factors that affect in vivo ribozyme (Rz) activity, a model system has been devised in Saccharomyces cerevisiae based on the inhibition of ADE1 gene expression. This gene was chosen because Rz action can be evaluated visually by the Red phenotype produced when the activity of the gene product is inhibited. Different plasmid constructs allowed the expression of the Rz either in cis or in trans with respect to ADE1. Rz-related inhibition of ADE1 expression was correlated with a Red phenotype and a diminution of ADE1 mRNA levels only when the Rz gene was linked 5' to ADE1. The presence of the expected 3' cleavage fragment was demonstrated using a technique combining RNA ligation and PCR. This yeast system and detection technique are suited to the investigation of general factors affecting Rz-catalyzed inhibition of gene expression under in vivo conditions.

  17. Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts.

    PubMed

    Tello, J; Cordero-Bueso, G; Aporta, I; Cabellos, J M; Arroyo, T

    2012-02-01

    Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. PCR-RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains' diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery-resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine-producing Appellation of Origin 'Vinos de Madrid' is shown. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  18. Influence of abiotic variables on culturable yeast diversity in two distinct Alpine glaciers.

    PubMed

    Turchetti, Benedetta; Goretti, Marta; Branda, Eva; Diolaiuti, Guglielmina; D'Agata, Carlo; Smiraglia, Claudio; Onofri, Andrea; Buzzini, Pietro

    2013-11-01

    The influence of some abiotic variables (pH, dry weight, organic carbon, nitrogen and phosphorous) on culturable yeast diversity in two distinct, but adjacent Alpine glaciers (Glacier du Géant, France, and Miage Glacier, Italy) was investigated. In all, 682 yeast strains were isolated and identified by D1/D2 and ITS sequencing as belonging to species of the genera Aureobasidium, Candida, Bulleromyces, Cryptococcus, Cystofilobasidium, Dioszegia, Guehomyces, Holtermanniella, Leucosporidiella, Mrakia, Mrakiella, Rhodotorula, Sporidiobolus, Sporobolomyces and Udenyomyces. Overall, the most represented genera were Cryptococcus (55% of isolates), Rhodotorula (17%) and Mrakia (10%). About 10% of strains, presumably belonging to new species (yet to be described), were preliminarily identified at the genus level. Principal component analysis (PCA) revealed that organic carbon, nitrogen and phosphorous are apparently mostly related to culturable yeast abundance and diversity. In this context, the hypothesis that the frequency of isolation of certain species may be correlated with some organic nutrients (with special emphasis for phosphorous) is discussed.

  19. Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential.

    PubMed

    Buzzini, Pietro; Branda, Eva; Goretti, Marta; Turchetti, Benedetta

    2012-11-01

    Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.

  20. A Survey of Essential Gene Function in the Yeast Cell Division Cycle

    PubMed Central

    Yu, Lisa; Castillo, Lourdes Peña; Mnaimneh, Sanie

    2006-01-01

    Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. PMID:16943325

  1. Nectar-living yeasts of a tropical host plant community: diversity and effects on community-wide floral nectar traits

    PubMed Central

    2017-01-01

    We characterize the diversity of nectar-living yeasts of a tropical host plant community at different hierarchical sampling levels, measure the associations between yeasts and nectariferous plants, and measure the effect of yeasts on nectar traits. Using a series of hierarchically nested sampling units, we extracted nectar from an assemblage of host plants that were representative of the diversity of life forms, flower shapes, and pollinator types in the tropical area of Yucatan, Mexico. Yeasts were isolated from single nectar samples; their DNA was identified, the yeast cell density was estimated, and the sugar composition and concentration of nectar were quantified using HPLC. In contrast to previous studies from temperate regions, the diversity of nectar-living yeasts in the plant community was characterized by a relatively high number of equally common species with low dominance. Analyses predict highly diverse nectar yeast communities in a relatively narrow range of tropical vegetation, suggesting that the diversity of yeasts will increase as the number of sampling units increases at the level of the species, genera, and botanical families of the hosts. Significant associations between specific yeast species and host plants were also detected; the interaction between yeasts and host plants impacted the effect of yeast cell density on nectar sugars. This study provides an overall picture of the diversity of nectar-living yeasts in tropical host plants and suggests that the key factor that affects the community-wide patterns of nectar traits is not nectar chemistry, but rather the type of yeasts interacting with host plants. PMID:28717591

  2. Nectar-living yeasts of a tropical host plant community: diversity and effects on community-wide floral nectar traits.

    PubMed

    Canto, Azucena; Herrera, Carlos M; Rodriguez, Rosalina

    2017-01-01

    We characterize the diversity of nectar-living yeasts of a tropical host plant community at different hierarchical sampling levels, measure the associations between yeasts and nectariferous plants, and measure the effect of yeasts on nectar traits. Using a series of hierarchically nested sampling units, we extracted nectar from an assemblage of host plants that were representative of the diversity of life forms, flower shapes, and pollinator types in the tropical area of Yucatan, Mexico. Yeasts were isolated from single nectar samples; their DNA was identified, the yeast cell density was estimated, and the sugar composition and concentration of nectar were quantified using HPLC. In contrast to previous studies from temperate regions, the diversity of nectar-living yeasts in the plant community was characterized by a relatively high number of equally common species with low dominance. Analyses predict highly diverse nectar yeast communities in a relatively narrow range of tropical vegetation, suggesting that the diversity of yeasts will increase as the number of sampling units increases at the level of the species, genera, and botanical families of the hosts. Significant associations between specific yeast species and host plants were also detected; the interaction between yeasts and host plants impacted the effect of yeast cell density on nectar sugars. This study provides an overall picture of the diversity of nectar-living yeasts in tropical host plants and suggests that the key factor that affects the community-wide patterns of nectar traits is not nectar chemistry, but rather the type of yeasts interacting with host plants.

  3. The longevity assurance homologue of yeast lag1 (Lass) gene family (review).

    PubMed

    Teufel, Andreas; Maass, Thorsten; Galle, Peter R; Malik, Nasir

    2009-02-01

    The Lass gene family contains a group of highly conserved genes that are found in eukaryotic species. The founding member, lag1, was discovered in a screen for yeast longevity genes. Subsequently, lag1 homologs were discovered in other organisms including six mammalian paralogs. All Lass genes encode a highly conserved Lag1 domain and many also have an additional Hox domain. Lass proteins are ceramide synthases and therefore are critical for ceramide biosynthesis. Ceramide synthase is also a critical enzyme in the sphingolipid biosynthetic pathway. As ceramide and sphingolipids are key intermediates in diverse cellular processes such as cell growth, apoptosis, and stress response and may also play a role in cancer development, the function of Lass proteins is of great interest. In this review, we summarize the state of knowledge regarding Lass protein structure, biological function, and their emerging role in cancer development.

  4. Black yeast diversity on creosoted railway sleepers changes with ambient climatic conditions.

    PubMed

    Gümral, Ramazan; Tümgör, Ayşegül; Saraçlı, Mehmet Ali; Yıldıran, Şinasi Taner; Ilkit, Macit; de Hoog, G Sybren

    2014-11-01

    The environmental isolation of opportunistic pathogenic black yeasts, which are responsible for a wide spectrum of human infections, is essential to understanding the ecology of clinical fungi. Extreme outdoor environments polluted with aromatic hydrocarbons support the growth of black yeasts in unlikely places, such as railway sleepers. However, there are limited data concerning the diversity of these fungi growing on polluted railway sleepers. In this investigation, we examined 845 railway sleeper samples, obtained from 11 Turkish cities representing altitudes from 25 to 1,893 m, and inoculated the samples onto mycological media for the isolation of black yeasts. Ninety-four samples (11.1 %) yielded positive results for black yeast, with creosoted oak sleepers having a significantly higher number of isolates than concrete sleepers (p < 0.05). Identification based on the ribosomal DNA (rDNA) internal transcribed spacer region revealed the highest prevalence of Exophiala phaeomuriformis, followed by Exophiala dermatitidis, Exophiala heteromorpha, Exophiala xenobiotica, and Exophiala crusticola. This study revealed that railway sleepers harboring black yeasts were predominantly (>75 %) populated with thermophilic species. We observed that altitude might have a significant effect on species diversity. Briefly, E. phaeomuriformis exhibited growth over a wide altitude range, from 30 to 1,893 m. In contrast, E. dermatitidis had a remarkable aversion to low altitudes and exhibited maximum growth at 1,285 m. In conclusion, we speculate that one can predict what species will be found on railway sleepers and their probability and that species diversity primarily depends on sleeper type and altitude height. We believe that this study can contribute new insights into the ecology of black yeasts on railway sleepers and the railway factors that influence their diversity.

  5. Yeast DNA sequences initiating gene expression in Escherichia coli.

    PubMed

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  6. Inferring Gene Family Histories in Yeast Identifies Lineage Specific Expansions

    PubMed Central

    Ames, Ryan M.; Money, Daniel; Lovell, Simon C.

    2014-01-01

    The complement of genes found in the genome is a balance between gene gain and gene loss. Knowledge of the specific genes that are gained and lost over evolutionary time allows an understanding of the evolution of biological functions. Here we use new evolutionary models to infer gene family histories across complete yeast genomes; these models allow us to estimate the relative genome-wide rates of gene birth, death, innovation and extinction (loss of an entire family) for the first time. We show that the rates of gene family evolution vary both between gene families and between species. We are also able to identify those families that have experienced rapid lineage specific expansion/contraction and show that these families are enriched for specific functions. Moreover, we find that families with specific functions are repeatedly expanded in multiple species, suggesting the presence of common adaptations and that these family expansions/contractions are not random. Additionally, we identify potential specialisations, unique to specific species, in the functions of lineage specific expanded families. These results suggest that an important mechanism in the evolution of genome content is the presence of lineage-specific gene family changes. PMID:24921666

  7. Aptamer-guided gene targeting in yeast and human cells

    PubMed Central

    Ruff, Patrick; Koh, Kyung Duk; Keskin, Havva; Pai, Rekha B.; Storici, Francesca

    2014-01-01

    Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting. PMID:24500205

  8. Functional conservation of the yeast and Arabidopsis RAD54-like genes.

    PubMed

    Klutstein, Michael; Shaked, Hezi; Sherman, Amir; Avivi-Ragolsky, Naomi; Shema, Efrat; Zenvirth, Drora; Levy, Avraham A; Simchen, Giora

    2008-04-01

    The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.

  9. Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias.

    PubMed

    Pando Bedriñana, R; Querol Simón, A; Suárez Valles, B

    2010-06-01

    This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22' and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of beta-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3' and 19') to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with beta-glucosidase activity are reported for the first time in autochthonous cider yeasts.

  10. Yeast diversity isolated from grape musts during spontaneous fermentation from a Brazilian winery.

    PubMed

    Bezerra-Bussoli, Carolina; Baffi, Milla Alves; Gomes, Eleni; Da-Silva, Roberto

    2013-09-01

    Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR-RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.

  11. Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach.

    PubMed

    Tantirungkij, Manee; Nasanit, Rujikan; Limtong, Savitree

    2015-09-01

    Endophytic microorganisms inhabit internal plant tissues in the host plant without causing any symptoms or negative effects. Although the diversity of endophytes has been evaluated by both culture-dependent and culture-independent methods, less information is available on yeast communities. Therefore, in this study a culture-independent method was used to examine endophytic yeasts associated with rice leaves based on the large subunit of ribosomal DNA using a semi-nested PCR technique. Sequence analysis indicated that the colonization frequency and the relative species frequency (RF) of endophytic yeast phylotypes were 0.41 and 0.06, respectively, and the majority of the yeast phylotypes were basidiomycetous yeasts. The phylotypes were designated as five known species (Cryptococcus victoriae, Debaryomyces hansenii, Debaryomyces vindobonensis, Meyerozyma guilliermondii and Pseudozyma antarctica), together with seventeen phylotypes closest to Candida metapsilosis, Cryp. foliicola, Cryp. laurentii, Pseudozyma abaconensis, Pseudozyma aphidis and Trichosporon asahii, among which some could be novel species. The most prevalent phylotypes were those closest to Cryp. foliicola (47.5 % RF) followed by D. hansenii (22.8 % RF) and P. antarctica (16.8 % RF). The presence of the phylotypes related to species known for their potential applications as biocontrol agents and plant growth promoting hormone producers suggests that they may have valuable applications. In addition, our findings revealed the occurrence of novel phylotypes at high frequency, which should encourage extensive studies to discover novel yeast species and to understand their roles in the rice leaves.

  12. Regulation of methanol utilisation pathway genes in yeasts

    PubMed Central

    Hartner, Franz S; Glieder, Anton

    2006-01-01

    Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts. PMID:17169150

  13. A large gene family in fission yeast encodes spore killers that subvert Mendel's law.

    PubMed

    Hu, Wen; Jiang, Zhao-Di; Suo, Fang; Zheng, Jin-Xin; He, Wan-Zhong; Du, Li-Lin

    2017-06-20

    Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve.

  14. Natural variation in non-coding regions underlying phenotypic diversity in budding yeast.

    PubMed

    Salinas, Francisco; de Boer, Carl G; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F; Martínez, Claudio; Cubillos, Francisco A

    2016-02-22

    Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype.

  15. Natural variation in non-coding regions underlying phenotypic diversity in budding yeast

    PubMed Central

    Salinas, Francisco; de Boer, Carl G.; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F.; Martínez, Claudio; Cubillos, Francisco A.

    2016-01-01

    Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype. PMID:26898953

  16. Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing.

    PubMed

    Wu, Hong; Watanabe, Tomoko; Araki, Yoshio; Kitagaki, Hiroshi; Akao, Takeshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2009-06-01

    Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.

  17. Genome-wide analysis of intraspecific transposon diversity in yeast

    PubMed Central

    2013-01-01

    Background In the model organism Saccharomyces cerevisiae, the transposable elements (TEs) consist of LTR (Long Terminal Repeat) retrotransposons called Ty elements belonging to five families, Ty1 to Ty5. They take the form of either full-length coding elements or non-coding solo-LTRs corresponding to remnants of former transposition events. Although the biological features of Ty elements have been studied in detail in S. cerevisiae and the Ty content of the reference strain (S288c) was accurately annotated, the Ty-related intra-specific diversity has not been closely investigated so far. Results In this study, we investigated the Ty contents of 41 available genomes of isolated S. cerevisiae strains of diverse geographical and ecological origins. The strains were compared in terms of the number of Ty copies, the content of the potential transpositionally active elements and the genomic insertion maps. The strain repertoires were also investigated in the closely related Ty1 and Ty2 families and subfamilies. Conclusions This is the first genome-wide analysis of the diversity associated to the Ty elements, carried out for a large set of S. cerevisiae strains. The results of the present analyses suggest that the current Ty-related polymorphism has resulted from multiple causes such as differences between strains, between Ty families and over time, in the recent transpositional activity of Ty elements. Some new Ty1 variants were also identified, and we have established that Ty1 variants have different patterns of distribution among strains, which further contributes to the strain diversity. PMID:23768249

  18. Pheromone-regulated genes required for yeast mating differentiation.

    PubMed

    Erdman, S; Lin, L; Malczynski, M; Snyder, M

    1998-02-09

    Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for beta-galactosidase (beta-gal) expression in the presence and absence of alpha factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: beta-gal and Fig2::beta-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

  19. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica.

    PubMed

    Vaz, Aline B M; Rosa, Luiz H; Vieira, Mariana L A; de Garcia, Virginia; Brandão, Luciana R; Teixeira, Lia C R S; Moliné, Martin; Libkind, Diego; van Broock, Maria; Rosa, Carlos A

    2011-07-01

    The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.

  20. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    PubMed Central

    Vaz, Aline B. M.; Rosa, Luiz H.; Vieira, Mariana L. A.; de Garcia, Virginia; Brandão, Luciana R.; Teixeira, Lia C. R. S.; Moliné, Martin; Libkind, Diego; van Broock, Maria; Rosa, Carlos A.

    2011-01-01

    The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates. PMID:24031709

  1. Generalized gene adjacencies, graph bandwidth, and clusters in yeast evolution.

    PubMed

    Zhu, Qian; Adam, Zaky; Choi, Vicky; Sankoff, David

    2009-01-01

    We present a parameterized definition of gene clusters that allows us to control the emphasis placed on conserved order within a cluster. Though motivated by biological rather than mathematical considerations, this parameter turns out to be closely related to the bandwidth parameter of a graph. Our focus will be on how this parameter affects the characteristics of clusters: how numerous they are, how large they are, how rearranged they are, and to what extent they are preserved from ancestor to descendant in a phylogenetic tree. We infer the latter property by dynamic programming optimization of the presence of individual edges at the ancestral nodes of the phylogeny. We apply our analysis to a set of genomes drawn from the Yeast Gene Order Browser.

  2. [Yeast diversity in Bulnesia retama and Larrea divaricata canopies and associated soils].

    PubMed

    Toro, M E; Oro, N P; Vega, A D; Maturano, Y P; Nally, M C; Fernandez, E; Pucheta, E; Vázquez, F

    2005-01-01

    Bush like vegetation dominates arid environments, and there is nutrients accumulation under shrub canopies and relatively unfertile soils between vegetal patches areas. Plants are one of the most common habitats for yeasts. There are many reports about yeasts inhabiting different plant components. Nevertheless, there are no reports about yeasts associated with Zygophyllaceae, an important shrub family of the Argentinean Province of Monte. The objective of this work was to analyzed yeast biodiversity of Bulnesia retama and Larrea divaricata canopies and associated soils, at Medanos Grandes of Caucete, San Juan, Argentina. Eighty seven (87) isolated yeasts were identified. From B. retama canopy and associated soil was observed a larger taxonomical diversity respect to L. divaricata. Nine (9) and ten (10) species were isolated from canopy and associated soil of B. retama, respectively. From L. divaricata canopy were 4 species and 3 species from its associated soil isolated. Identified genera were: Candida, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Sporidiobolus and Pichia. Fourteen (14) species were found at all microenvironments.

  3. Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.

    PubMed

    Daniel, Jewel A; Keyes, Brice E; Ng, Yvonne P Y; Freeman, C Onyi; Burke, Daniel J

    2006-01-01

    The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

  4. A Genetic Screen for Fission Yeast Gene Deletion Mutants Exhibiting Hypersensitivity to Latrunculin A

    PubMed Central

    Asadi, Farzad; Michalski, Dorothy; Karagiannis, Jim

    2016-01-01

    Fission yeast cells treated with low doses of the actin depolymerizing drug, latrunculin A (LatA), delay entry into mitosis via a mechanism that is dependent on both the Clp1p and Rad24p proteins. During this delay, cells remain in a cytokinesis-competent state that is characterized by continuous repair and/or reestablishment of the actomyosin ring. In this manner, cells ensure the faithful completion of the preceding cytokinesis in response to perturbation of the cell division machinery. To uncover other genes with a role in this response, or simply genes with roles in adapting to LatA-induced stress, we carried out a genome-wide screen and identified a group of 38 gene deletion mutants that are hyper-sensitive to the drug. As expected, we found genes affecting cytokinesis and/or the actin cytoskeleton within this set (ain1, acp2, imp2). We also identified genes with roles in histone modification (tra1, ngg1), intracellular transport (apl5, aps3), and glucose-mediated signaling (git3, git5, git11, pka1, cgs2). Importantly, while the identified gene deletion mutants are prone to cytokinesis failure in the presence of LatA, they are nevertheless fully capable of cell division in the absence of the drug. These results indicate that fission yeast cells make use of a diverse set of regulatory modules to counter abnormal cytoskeletal perturbations, and furthermore, that these modules act redundantly to ensure cell survival and proliferation. PMID:27466272

  5. The molecular characterization of new types of Saccharomyces cerevisiae×S. kudriavzevii hybrid yeasts unveils a high genetic diversity.

    PubMed

    Peris, David; Belloch, Carmela; Lopandić, Ksenija; Álvarez-Pérez, José Manuel; Querol, Amparo; Barrio, Eladio

    2012-02-01

    New double- and triple-hybrid Saccharomyces yeasts were characterized using PCR-restriction fragment length polymorphism of 35 nuclear genes, located on different chromosome arms, and the sequencing of one nuclear and one mitochondrial gene. Most of these new hybrids were originally isolated from fermentations; however, two of them correspond to clinical and dietary supplement isolates. This is the first time that the presence of double-hybrid S. cerevisiae×S. kudriavzevii in non-fermentative substrates has been reported and investigated. Phylogenetic analysis of the MET6 nuclear gene confirmed the double or triple parental origin of the new hybrids. Restriction analysis of gene regions in these hybrids revealed a high diversity of genome types. From these molecular characterizations, a reduction of the S. kudriavzevii fraction of the hybrid genomes is observed in most hybrids. Mitochondrial inheritance in hybrids was deduced from the analysis of mitochondrial COX2 gene sequences, which showed that most hybrids received the mitochondrial genome from the S. kudriavzevii parent. However, two strains inherited a S. cerevisiae COX2, being the first report of S. cerevisiae×S. kudriavzevii hybrids with S. cerevisiae mitochondrial genomes. These two strains are those showing a higher S. kudriavzevii nuclear genome reduction, especially in the wine hybrid AMH. This may be due to the release of selective pressures acting on the other hybrids to maintain kudriavzevii mitochondria-interacting genes.

  6. Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

    SciTech Connect

    Mendez, M.J.; Abderrahim, H.; Noguchi, M.

    1995-03-20

    With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (V{sub H}) genes, the major diversity (D) gene cluster, the joining (J{sub H}) genes, the intronic enhancer (E{sub H}), and the constant region genes, mu (C{mu}) and delta (C{delta}). Two IgK locus-derived YACs each contain three variable (V{kappa}) genes, the joining (J{kappa}) region, the intronic enhancer (E{kappa}), the constant gene (C{kappa}), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form. 78 refs., 7 figs.

  7. The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression.

    PubMed

    Swinkels, B W; van Ooyen, A J; Bonekamp, F J

    Several different yeast species have been developed into systems for efficient heterologous gene expression. In this paper we review foreign gene expression in the dairy yeast Kluyveromyces lactis. This yeast presents several advantageous properties in comparison to other yeast species. These include its impressive secretory capacities, its excellent fermentation characteristics on large scale, its food grade status and the availability of both episomal and integrative expression vectors. Moreover, in contrast to the methylotrophic yeasts that are frequently used for the expression of foreign genes, K. lactis does not require explosion-proof fermentation equipment. Here, we present an overview of the available tools for heterologous gene expression in K. lactis (available promoters, vector systems, etc). Also, the production of prochymosin, human serum albumin and pancreatic phospholipase by K. lactis is discussed in more detail, and used to rate the achievements of K. lactis with respect to other micro-organisms in which these proteins have been produced.

  8. A yeast functional screen predicts new candidate ALS disease genes

    PubMed Central

    Couthouis, Julien; Hart, Michael P.; Shorter, James; DeJesus-Hernandez, Mariely; Erion, Renske; Oristano, Rachel; Liu, Annie X.; Ramos, Daniel; Jethava, Niti; Hosangadi, Divya; Epstein, James; Chiang, Ashley; Diaz, Zamia; Nakaya, Tadashi; Ibrahim, Fadia; Kim, Hyung-Jun; Solski, Jennifer A.; Williams, Kelly L.; Mojsilovic-Petrovic, Jelena; Ingre, Caroline; Boylan, Kevin; Graff-Radford, Neill R.; Dickson, Dennis W.; Clay-Falcone, Dana; Elman, Lauren; McCluskey, Leo; Greene, Robert; Kalb, Robert G.; Lee, Virginia M.-Y.; Trojanowski, John Q.; Ludolph, Albert; Robberecht, Wim; Andersen, Peter M.; Nicholson, Garth A.; Blair, Ian P.; King, Oliver D.; Bonini, Nancy M.; Van Deerlin, Vivianna; Rademakers, Rosa; Mourelatos, Zissimos; Gitler, Aaron D.

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. PMID:22065782

  9. [CITRULLINUREIDASE GENE DIVERSITY IN THE GENUS FRANCISELLA].

    PubMed

    Timofeev, V S; Bakhteeva, I V; Pavlov, V M; Mokrievich, A N

    2015-01-01

    This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

  10. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

    PubMed

    Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

    2011-09-01

    The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.

  11. A yeast transcription system for the 5S rRNA gene.

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin. Images PMID:7145700

  12. An eYFP reporter gene for the yeast two-hybrid system.

    PubMed

    Damon, Coralie; Boxus, Mathieu; Twizere, Jean-Claude; Portetelle, Daniel; Vandenbol, Micheline

    2013-02-01

    The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.

  13. Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes.

    PubMed

    Brandão, Luciana R; Libkind, Diego; Vaz, Aline B M; Espírito Santo, Lília C; Moliné, Martín; de García, Virginia; van Broock, Maria; Rosa, Carlos A

    2011-04-01

    Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with high transparency, surrounded by well-developed forests and located at San Carlos de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we characterized yeast distribution and diversity along a south-to-north transect and established a relationship between the ability to produce photoprotective compounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of yeast at different collection points. Subsurface water samples were filtered for yeast isolation. Total yeast counts ranged between 22 and 141 CFU L(-1) , and the highest values corresponded to the most impacted sites. Littoral sites had a low proportion of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably a result of the high transparency of the water and the increased UV exposure. The yeast community from NH Lake showed a high species richness and a uniform distribution of taxa between pelagic and border collection points. Yeasts were identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and Cryptococcus victoriae were the most frequently found species, representing 14.4% and 13.6% of the total yeast isolates, respectively. Most of the yeast isolates demonstrated at least one extracellular enzymatic activity (mainly cellulase and lipase activities), which suggested that these microorganisms are metabolically active in the lake. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Yeast Methylotrophy: Metabolism, Gene Regulation and Peroxisome Homeostasis

    PubMed Central

    Yurimoto, Hiroya; Oku, Masahide; Sakai, Yasuyoshi

    2011-01-01

    Eukaryotic methylotrophs, which are able to obtain all the carbon and energy needed for growth from methanol, are restricted to a limited number of yeast species. When these yeasts are grown on methanol as the sole carbon and energy source, the enzymes involved in methanol metabolism are strongly induced, and the membrane-bound organelles, peroxisomes, which contain key enzymes of methanol metabolism, proliferate massively. These features have made methylotrophic yeasts attractive hosts for the production of heterologous proteins and useful model organisms for the study of peroxisome biogenesis and degradation. In this paper, we describe recent insights into the molecular basis of yeast methylotrophy. PMID:21754936

  15. Genetic diversity of FLO1 and FLO5 genes in wine flocculent Saccharomyces cerevisiae strains.

    PubMed

    Tofalo, Rosanna; Perpetuini, Giorgia; Di Gianvito, Paola; Schirone, Maria; Corsetti, Aldo; Suzzi, Giovanna

    2014-11-17

    Twenty-eight flocculent wine strains were tested for adhesion and flocculation phenotypic variability. Moreover, the expression patterns of the main genes involved in flocculation (FLO1, FLO5 and FLO8) were studied both in synthetic medium and in presence of ethanol stress. Molecular identification and typing were achieved by PCR-RFLP of the 5.8S ITS rRNA region and microsatellite PCR fingerprinting, respectively. All isolates belong to Saccharomyces cerevisiae species. The analysis of microsatellites highlighted the intraspecific genetic diversity of flocculent wine S. cerevisiae strains allowing obtaining strain-specific profiles. Moreover, strains were characterized on the basis of adhesive properties. A wide biodiversity was observed even if none of the tested strains were able to form biofilms (or 'mats'), or to adhere to polystyrene. Moreover, genetic diversity of FLO1 and FLO5 flocculating genes was determined by PCR. Genetic diversity was detected for both genes, but a relationship with the flocculation degree was not found. So, the expression patterns of FLO1, FLO5 and FLO8 genes was investigated in a synthetic medium and a relationship between the expression of FLO5 gene and the flocculation capacity was established. To study the expression of FLO1, FLO5 and FLO8 genes in floc formation and ethanol stress resistance qRT-PCR was carried out and also in this case strains with flocculent capacity showed higher levels of FLO5 gene expression. This study confirmed the diversity of flocculation phenotype and genotype in wine yeasts. Moreover, the importance of FLO5 gene in development of high flocculent characteristic of wine yeasts was highlighted. The obtained collection of S. cerevisiae flocculent wine strains could be useful to study the relationship between the genetic variation and flocculation phenotype in wine yeasts.

  16. Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity.

    PubMed

    Sporn, Zachary A; Hines, Justin K

    2015-01-01

    Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

  17. Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

    PubMed

    Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E

    2015-03-01

    Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately.

  18. An estradiol-inducible promoter enables fast, graduated control of gene expression in fission yeast.

    PubMed

    Ohira, Makoto J; Hendrickson, David G; Scott McIsaac, R; Rhind, Nicholas

    2017-08-01

    The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose-response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3 EV, turns on quickly, can reach a maximal induction of 20-fold, and exhibits a linear dose response over its entire induction range, with few off-target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Quantitative analysis of wine yeast gene expression profiles under winemaking conditions.

    PubMed

    Varela, Cristian; Cárdenas, Javier; Melo, Francisco; Agosin, Eduardo

    2005-04-15

    Wine fermentation is a dynamic and complex process in which the yeast cell is subjected to multiple stress conditions. A successful adaptation involves changes in gene expression profiles where a large number of genes are up- or downregulated. Functional genomic approaches are commonly used to obtain global gene expression profiles, thereby providing a comprehensive view of yeast physiology. We used SAGE to quantify gene expression profiles in an industrial strain of Saccharomyces cerevisiae under winemaking conditions. The transcriptome of wine yeast was analysed at three stages during the fermentation process, mid-exponential phase, and early- and late-stationary phases. Upon correlation with the yeast genome, we found three classes of transcripts: (a) sequences that corresponded to ORFs; (b) expressed sequences from intergenic regions; and (c) messengers that did not match the published reference yeast genome. In all fermentation phases studied, the most highly expressed genes related to energy production and stress response. For many pathways, including glycolysis, different transcript levels were observed during each phase. Different isoenzymes, including hexose transporters (HXT), were differentially induced, depending on the growth phase. About 10% of transcripts matched non-annotated ORF regions within the yeast genome and could correspond to small novel genes originally omitted in the first gene annotation effort. Up to 22% of transcripts, particularly at late-stationary phase, did not match any known location within the genome. As the available reference yeast genome was obtained from a laboratory strain, these expressed sequences could represent genes only expressed by an industrial yeast strain. Further studies are necessary to identify the role of these potential genes during wine fermentation.

  20. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

    PubMed Central

    2012-01-01

    Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures. PMID:23131126

  1. Matrix factorization-based data fusion for gene function prediction in baker's yeast and slime mold.

    PubMed

    Zitnik, Marinka; Zupan, Blaž

    2014-01-01

    The development of effective methods for the characterization of gene functions that are able to combine diverse data sources in a sound and easily-extendible way is an important goal in computational biology. We have previously developed a general matrix factorization-based data fusion approach for gene function prediction. In this manuscript, we show that this data fusion approach can be applied to gene function prediction and that it can fuse various heterogeneous data sources, such as gene expression profiles, known protein annotations, interaction and literature data. The fusion is achieved by simultaneous matrix tri-factorization that shares matrix factors between sources. We demonstrate the effectiveness of the approach by evaluating its performance on predicting ontological annotations in slime mold D. discoideum and on recognizing proteins of baker's yeast S. cerevisiae that participate in the ribosome or are located in the cell membrane. Our approach achieves predictive performance comparable to that of the state-of-the-art kernel-based data fusion, but requires fewer data preprocessing steps.

  2. Large-Scale Analysis of Yeast Filamentous Growth by Systematic Gene Disruption and Overexpression

    PubMed Central

    Jin, Rui; Dobry, Craig J.; McCown, Phillip J.

    2008-01-01

    Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Σ1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth. PMID:17989363

  3. Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments.

    PubMed

    Choudhary, Jairam; Singh, Surender; Nain, Lata

    2017-03-01

    Lignocellulosic biomass, a promising renewable energy source, can be used for the production of second generation bioethanol. Simultaneous saccharification and fermentation (SSF), the process which alleviates the problem of separate hydrolysis and fermentation (SHF), requires thermotolerant ethanologenic yeast for bioethanol production. Therefore, ten yeast strains isolated from diverse sources, belonging to various genera like Saccharomyces, Candida, Pichia and Wickerhamomyces were evaluated for their thermotolerance, sugar utilization pattern, inhibitor tolerance and ethanol production potential with glucose, xylose and alkali pretreated paddy straw. All the tested strains were found to be thermotolerant, capable of significant growth at 40°C. Candida tropicalis Y6 was capable of utilizing a wide range of sugars as compared with other yeast isolates. Strains of Candida showed better inhibitor tolerance as compared to Saccharomyces and Pichia strains and exhibited only 5.1-18.8% and 4.7-7.9% reduction in growth with furfural and 5-hydroxymethyl furfural, respectively. Saccharomyces cerevisiae JRC6, isolated from distillery waste, produced ethanol with 88.3% and 89.1% theoretical efficiency at 40°C and 42°C, respectively, from glucose. This strain also produced significantly higher amount of ethanol (3.8 g/L) with better fermentation efficiency (87.9%) from alkali pretreated paddy straw at 40°C, as compared with the other yeast strains. Therefore, S. cerevisiae JRC6, based on its ability to ferment sugars at a higher temperature, can be a promising candidate for production of ethanol from lignocellulosic biomass via SSF process.

  4. Chromosomal Integration and Expression of Two Bacterial α-Acetolactate Decarboxylase Genes in Brewer's Yeast

    PubMed Central

    Blomqvist, K.; Suihko, M.-L.; Knowles, J.; Penttilä, M.

    1991-01-01

    A bacterial gene encoding α-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the α-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the α-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer. Images PMID:16348559

  5. Discrete dynamical system modelling for gene regulatory networks of 5-hydroxymethylfural tolerance for ethanologenic yeast

    USDA-ARS?s Scientific Manuscript database

    Composed of linear difference equations, a discrete dynamic system model was designed to reconstruct transcriptional regulations in gene regulatory networks in response to 5-hydroxymethylfurfural, a bioethanol conversion inhibitor for ethanologenic yeast Saccharomyces cerevisiae. The modeling aims ...

  6. Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

    PubMed Central

    Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

    2006-01-01

    During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994

  7. The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments.

    PubMed

    Masneuf-Pomarede, Isabelle; Juquin, Elodie; Miot-Sertier, Cécile; Renault, Philippe; Laizet, Yec'han; Salin, Franck; Alexandre, Hervé; Capozzi, Vittorio; Cocolin, Luca; Colonna-Ceccaldi, Benoit; Englezos, Vasileios; Girard, Patrick; Gonzalez, Beatriz; Lucas, Patrick; Mas, Albert; Nisiotou, Aspasia; Sipiczki, Matthias; Spano, Giuseppe; Tassou, Chrysoula; Bely, Marina; Albertin, Warren

    2015-08-01

    The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology.

    PubMed

    Gupta, Rani; Kumari, Arti; Syal, Poonam; Singh, Yogesh

    2015-01-01

    Lipase catalyzes hydrolysis of fats in lipid water interphase and perform variety of biotransformation reactions under micro aqueous conditions. The major sources include microbial lipases; among these yeast and fungal lipases are of special interest because they can carry out various stereoselective reactions. These lipases are highly diverse and are categorized into three classes on the basis of oxyanion hole: GX, GGGX and Y. The detailed phylogenetic analysis showed that GX family is more diverse than GGGX and Y family. Sequence and structural comparisons revealed that lipases are conserved only in the signature sequence region. Their characteristic structural determinants viz. lid, binding pocket and oxyanion hole are hotspots for mutagenesis. Few examples are cited in this review to highlight the multidisciplinary approaches for designing novel enzyme variants with improved thermo stability and substrate specificity. In addition, we present a brief account on biotechnological applications of lipases. Lipases have also gained attention as virulence factors, therefore, we surveyed the role of lipases in yeast physiology related to colonization, adhesion, biofilm formation and pathogenesis. The new genomic era has opened numerous possibilities to genetically manipulate lipases for food, fuel and pharmaceuticals.

  9. Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

    PubMed Central

    Larkin, J C; Woolford, J L

    1983-01-01

    Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed. Images PMID:6338478

  10. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast.

    PubMed

    Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

    2013-08-01

    Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. © 2013 The Authors. Aging Cell published by John Wiley & Sons Ltd and the Anatomical Society.

  11. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast

    PubMed Central

    Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

    2013-01-01

    Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. PMID:23551936

  12. A large gene family in fission yeast encodes spore killers that subvert Mendel’s law

    PubMed Central

    Hu, Wen; Jiang, Zhao-Di; Suo, Fang; Zheng, Jin-Xin; He, Wan-Zhong; Du, Li-Lin

    2017-01-01

    Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve. DOI: http://dx.doi.org/10.7554/eLife.26057.001 PMID:28631610

  13. Enhancement of the Gene Targeting Efficiency of Non-Conventional Yeasts by Increasing Genetic Redundancy

    PubMed Central

    Chen, Zao; Sun, Hongbing; Li, Pengfei; He, Ning; Zhu, Taicheng; Li, Yin

    2013-01-01

    In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting. PMID:23505447

  14. Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

    PubMed

    Silverman, Andrew M; Qiao, Kangjian; Xu, Peng; Stephanopoulos, Gregory

    2016-04-01

    Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH

  15. Origins and kinetic consequences of diversity in Sup35 yeast prion fibers.

    PubMed

    DePace, Angela H; Weissman, Jonathan S

    2002-05-01

    A remarkable feature of prions is that infectious particles composed of the same prion protein can give rise to different phenotypes. This strain phenomenon suggests that a single prion protein can adopt multiple infectious conformations. Here we use a novel single fiber growth assay to examine the heterogeneity of amyloid fibers formed by the yeast Sup35 prion protein. Sup35 spontaneously forms multiple, distinct and faithfully propagating fiber types, which differ dramatically both in their degrees of polarity and overall growth rates. Both in terms of the number of distinct self-propagating fiber types, as well as the ability of these differences to dictate the rate of prion growth, this diversity is well suited to account for the range of prion strain phenotypes observed in vivo.

  16. Yeast Pho85 kinase is required for proper gene expression during the diauxic shift.

    PubMed

    Nishizawa, Masafumi; Katou, Yuki; Shirahige, Katsuhiko; Toh-e, Akio

    2004-08-01

    The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.

  17. Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

    PubMed Central

    Jespersen, Lene; Cesar, Lene B.; Meaden, Philip G.; Jakobsen, Mogens

    1999-01-01

    Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure. PMID:9925567

  18. A series of promoters for constitutive expression of heterologous genes in fission yeast.

    PubMed

    Matsuyama, Akihisa; Shirai, Atsuko; Yoshida, Minoru

    2008-05-01

    Inducible/repressible promoters are useful for the maintenance of toxic genes or timely expression. For ectopic expression of cloned genes in the fission yeast Schizosaccharomyces pombe, the thiamine-regulatable nmt1 promoter has been widely used, since the transcriptional activity of this promoter can be controlled by thiamine. However, this property sometimes limits a certain type of research, since the expression inevitably requires cells to be cultivated under the conditions that induce promoter activation. To allow constitutive expression of heterologous genes, we cloned three promoters of cam1+, tif51+ and ef1a-c+. Construction of a series of vectors comprising these promoters and their introduction into the fission yeast cells demonstrated that the activity was different among these promoters but was not affected by cultured media commonly used in fission yeast. Therefore, a promoter with appropriate strength would be selectable from these promoters, depending on the genes to be expressed.

  19. Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough

    PubMed Central

    Holmes, Rebecca K.; Tuck, Alex C.; Zhu, Chenchen; Dunn-Davies, Hywel R.; Kudla, Grzegorz; Clauder-Munster, Sandra; Granneman, Sander; Steinmetz, Lars M.; Guthrie, Christine; Tollervey, David

    2015-01-01

    Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3’ end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation. Tiling arrays and RNAPII mapping data revealed 3’ extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression. PMID:26694144

  20. Cloning, sequence analysis and yeast expression of the egl1 gene from Trichoderma longibrachiatum.

    PubMed

    González, R; Ramón, D; Pérez-González, J A

    1992-12-01

    A gene (egl1) encoding an endoglucanase (EGL1) from Trichoderma longibrachiatum has been cloned and sequenced. This gene, homologous to the T. reesei egl1 gene, differs from it in the length of the introns (particularly the first one) and encoded protein. A cDNA fragment obtained by the rapid amplification of cDNA ends method, which takes advantage of the polymerase chain reaction, has been expressed in yeast under control of the cyc-gal inducible promoter and yeast clones able to secrete active enzyme have been obtained.

  1. Segregation of yeast polymorphic STA genes in meiotic recombinants and analysis of glucoamylase production.

    PubMed

    Balogh, I; Maráz, A

    1996-12-01

    Hybrid yeast strains were constructed using haploid Saccharomyces cerevisiae and Saccharomyces cerevisiae var. diastaticus strains to get haploid meiotic recombinants having more than one copy of STA1, STA2, and STA3 genes. STA genes were localized on the chromosomes by pulsed field gel electrophoresis. Working gene dosage effects were found among STA genes in liquid starch medium, indicating low levels of glucose repression. Growth of strains, however, was not influenced by their STA copy number.

  2. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  3. Occurrence and diversity of yeasts in the mid-atlantic ridge hydrothermal fields near the Azores Archipelago.

    PubMed

    Gadanho, Mário; Sampaio, José Paulo

    2005-10-01

    The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new

  4. The Insertion Green Monster (iGM) Method for Expression of Multiple Exogenous Genes in Yeast

    PubMed Central

    Labunskyy, Vyacheslav M.; Suzuki, Yo; Hanly, Timothy J.; Murao, Ayako; Roth, Frederick P.; Gladyshev, Vadim N.

    2014-01-01

    Being a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains expressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the “insertion Green Monster” (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof of principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci. PMID:24776987

  5. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  6. [HSM6 gene is identical to PSY4 gene in Saccharomyces cerevisiae yeasts].

    PubMed

    Fedorov, D V; Koval'tsova, S V; Evstukhina, T A; Peshekhonov, V T; Chernenkov, A Iu; Korolev, V G

    2013-03-01

    Previously, we isolated mutant yeasts Saccharomyces cerevisiae with an increased rate of spontaneous mutagenesis. Here, we studied the properties of HSM6 gene, the hsm6-1 mutation of which increased the frequency of UV-induced mutagenesis and decreased the level of UV-induced mitotic crossover at the centromere gene region, ADE2. HSM6 gene was mapped on the left arm of chromosome 11 in the region where the PSY4 gene is located. The epistatic analysis has shown that the hsm6-1 mutation represents an allele of PSY4 gene. Sequencing of hsm6-1 mutant allele has revealed a frameshift mutation, which caused the substitution of Lys218Glu and the generation of a stop codon in the next position. The interactions of hsm6-1 and rad52 mutations were epistatic. Our data show that the PSY4 gene plays a key role in the regulation of cell withdrawal from checkpoint induced by DNA disturbances.

  7. Seasonal and plant-dependent variations in diversity, abundance and stress tolerance of epiphytic yeasts in desert habitats.

    PubMed

    Abu-Ghosh, Said; Droby, Samir; Korine, Carmi

    2014-08-01

    We studied the epiphytic yeast species of the plants of the Negev Desert and the Dead Sea region, Israel, which are considered one of the most extreme hyper-arid lands in the world. For this purpose, we developed isolation protocols; we performed morphological, cultural and molecular identification tests and compared yeast diversity between the locations and the plants. The composition of the yeast populations present in the study's plants underwent seasonal fluctuations, whereas differences in community compositions were significant within sites. The maximum number of species of yeast occurred in autumn and Cryptococcus spp. were predominant year round. The isolated yeast strains showed an unusual tolerance to extreme growth conditions, such as high temperatures (up to 72% viability at 50°C), lethal hydrogen peroxide and NaCl concentrations. These results suggest that epiphytic yeasts inhabit the plants of the Dead Sea region and the Negev Desert have a community structure that is unique to the plant species and have a high tolerance to the harsh conditions that enables them to adapt to an arid ecosystem.

  8. Gene regulatory changes in yeast during life extension by nutrient limitation.

    PubMed

    Wang, Jinqing; Jiang, James C; Jazwinski, S Michal

    2010-08-01

    Genetic analyses aimed at identification of the pathways and downstream effectors of calorie restriction (CR) in the yeast Saccharomyces cerevisiae suggest the importance of central metabolism for the extension of replicative life span by CR. However, the limited gene expression studies to date are not informative, because they have been conducted using cells grown in batch culture which markedly departs from the conditions under which yeasts are grown during life span determinations. In this study, we have examined the gene expression changes that occur during either glucose limitation or elimination of nonessential-amino acids, both of which enhance yeast longevity, culturing cells in a chemostat at equilibrium, which closely mimics conditions they encounter during life span determinations. Expression of 59 genes was examined quantitatively by real-time, reverse transcriptase polymerase chain reaction (qRT-PCR), and the physiological state of the cultures was monitored. Extensive gene expression changes were detected, some of which were common to both CR regimes. The most striking of these was the induction of tricarboxylic acid (TCA) cycle and retrograde response target genes, which appears to be at least partially due to the up-regulation of the HAP4 gene. These gene regulatory events portend an increase in the generation of biosynthetic intermediates necessary for the production of daughter cells, which is the measure of yeast replicative life span. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  9. Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

    PubMed Central

    Panavas, Tadas; Serviene, Elena; Brasher, Jeremy; Nagy, Peter D.

    2005-01-01

    Viruses are devastating pathogens of humans, animals, and plants. To further our understanding of how viruses use the resources of infected cells, we systematically tested the yeast single-gene-knockout library for the effect of each host gene on the replication of tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins, and other compounds and in protein targeting/transport. Comparison with published genome-wide screens reveals that the replication of TBSV and brome mosaic virus (BMV), which belongs to a different supergroup among plus-strand RNA viruses, is affected by vastly different yeast genes. Moreover, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related α-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This finding suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may suggest connections between chronic diseases and prior virus infection. PMID:15883361

  10. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

    PubMed Central

    Schild, D; Brake, A J; Kiefer, M C; Young, D; Barr, P J

    1990-01-01

    Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme. Images PMID:2183217

  11. Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

    PubMed

    Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

    2014-05-16

    Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

  12. Yeast diversity associated with invasive Dendroctonus valens killing Pinus tabuliformis in China using culturing and molecular methods.

    PubMed

    Lou, Qiao-Zhe; Lu, Min; Sun, Jiang-Hua

    2014-08-01

    Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are

  13. Distribution of the trehalase activation response and the regulatory trehalase gene among yeast species.

    PubMed

    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.

  14. Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

    PubMed Central

    Morlino, Giovanni B.; Tizzani, Lorenza; Fleer, Reinhard; Frontali, Laura; Bianchi, Michele M.

    1999-01-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system. PMID:10543790

  15. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    PubMed

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  16. Large-scale analysis of the yeast genome by transposon tagging and gene disruption.

    PubMed

    Ross-Macdonald, P; Coelho, P S; Roemer, T; Agarwal, S; Kumar, A; Jansen, R; Cheung, K H; Sheehan, A; Symoniatis, D; Umansky, L; Heidtman, M; Nelson, F K; Iwasaki, H; Hager, K; Gerstein, M; Miller, P; Roeder, G S; Snyder, M

    1999-11-25

    Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

  17. Diversity of Saccharomyces cerevisiae yeasts associated to spontaneously fermenting grapes from an Italian "heroic vine-growing area".

    PubMed

    Capece, A; Romaniello, R; Siesto, G; Romano, P

    2012-09-01

    The main aim of this work was to analyse the diversity of wild Saccharomyces cerevisiae isolated from spontaneous fermentations of grapes collected from heroic vine-growing area. A first screening based on several technological traits was used to select 39 strains among 132 isolates. By using three molecular typing techniques (evaluation of cell wall gene polymorphisms, mtDNA restriction analysis, inter-delta amplification analysis) a significant genetic variability was found. The analysis of principal aromatic compounds produced during inoculated fermentation of two grape musts demonstrated the strain impact on wine flavour and a significant influence of grape must on strain metabolic behavior. One selected strain was used in fermentation at cellar level and the analysis of inter-delta region on yeast colonies isolated during the process revealed the high-implantation ability of this strain. The obtained results demonstrate the usefulness of different molecular and technological markers for the evaluation of natural biodiversity among S. cerevisiae strains. This study represents an essential step towards the exploitation and the preservation of biodiversity of strains isolated from heroic vine-growing area. Selected S. cerevisiae strains could represent starter cultures available for winemakers addressed to production of quality premium wines maintaining differential properties of their own area.

  18. Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Hegemann, Johannes H; Heick, Sven Boris

    2011-01-01

    Gene inactivation is an essential step in the molecular dissection of gene function. In the yeast Saccharomyces cerevisiae, many tools for gene disruption are available. Gene disruption cassettes comprising completely heterologous marker genes flanked by short DNA segments homologous to the regions to the left and right of the gene to be deleted mediate highly efficient one-step gene disruption events. Routinely, in more than 50% of analyzed clones, the marker cassette is integrated in the targeted location. The inclusion of loxP sites flanking the disruption marker gene allows sequence-specific Cre recombinase-mediated marker rescue so that the marker can be reused to disrupt another gene. Here, we describe a comprehensive toolbox for multiple gene disruptions comprising a set of seven heterologous marker genes including four dominant resistance markers for gene disruption, plus a set of Cre expression plasmids carrying eight different selection markers, four of them dominant.

  19. The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

    PubMed

    Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

    2011-03-01

    Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

  20. A densely overlapping gene fragmentation approach improves yeast two-hybrid screens for Plasmodium falciparum proteins.

    PubMed

    Brown, Hakeenah F; Wang, Ling; Khadka, Sudip; Fields, Stanley; LaCount, Douglas J

    2011-01-01

    Use of the yeast two-hybrid assay to study Plasmodium falciparum protein-protein interactions is limited by poor expression of P. falciparum genes in yeast and lack of easily implemented assays to confirm the results. We report here two methods to create gene fragments - random fragmentation by partial DNAse I digestion and generation of densely overlapping fragments by PCR - that enable most portions of P. falciparum genes to be expressed and screened in the yeast two-hybrid assay. The PCR-based method is less technically challenging and facilitates fine-scale mapping of protein interaction domains. Both approaches revealed a putative interaction between PfMyb2 (PF10_0327) and PFC0365w. We developed new plasmids to express the proteins in wheat germ extracts and confirmed the interaction in both the split-luciferase assay and in co-purification experiments with glutathione-S-transferase and HA-tagged proteins. The combination of improved yeast two-hybrid screening approaches and convenient systems to validate interactions enhances the utility of yeast two-hybrid assays for P. falciparum.

  1. Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Scientific note.

    PubMed

    Deng, X X; Ho, N W

    1990-01-01

    D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeast can ferment xylose and xylulose more than twice as fast as the parent yeast.

  2. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

  3. Yeasts from native Brazilian Cerrado plants: Occurrence, diversity and use in the biocontrol of citrus green mould.

    PubMed

    Sperandio, Eugenio Miranda; do Vale, Helson Mario Martins; Moreira, Geisianny Augusta Monteiro

    2015-11-01

    Yeasts are some of the most important postharvest biocontrol agents. Postharvest oranges frequently deteriorate due to green mould (Penicillium digitatum), which causes significant losses. The aims of this study were to determine the composition and diversity of yeasts on plants of the Brazilian Cerrado and to explore their potential for inhibiting citrus green mould. Leaves and fruit of Byrsonima crassifolia and Eugenia dysenterica were collected from Cerrado conservation areas, and thirty-five yeasts were isolated and identified by sequencing the D1-D2 domain of the rDNA large subunit (26S). The isolates represented the Aureobasidium, Meyerozyma, Candida, and Pichia genera. Three isolates identified as Aureobasidium pullulans exhibited potential for the control of P. digitatum in both in vitro and in vivo tests; these isolates reduced the incidence of disease and increased the storage time of fruit. Aureobasidium. pullulans has immense potential for the biological control of filamentous fungi.

  4. Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast

    PubMed Central

    Bergkessel, Megan; Whitworth, Gregg B.; Guthrie, Christine

    2011-01-01

    Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast. PMID:21697354

  5. Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast.

    PubMed

    Bergkessel, Megan; Whitworth, Gregg B; Guthrie, Christine

    2011-08-01

    Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast.

  6. Introduction of the yeast DNA repair gene PHR1 into normal and xeroderma pigmentosum human cells

    SciTech Connect

    Whyte, D.B.

    1988-01-01

    The goal of the work described herein is to determine how UV light kills and mutates human cells. Specifically, the hypothesis to be tested states that the major cause of cell death is the cyclobutane dimer. The yeast (S. cerevisiae) enzyme photolyase provides an elegant means of dissecting the biological effects of the two lesions. Photolyase, the product of the PHR1 gene, catalyzes the visible light-dependent reversal of cyclobutane pyrimidine dimers. Introducing the gene for photolyase into human cells, which do not have a functional photoreactivation mechanism, should allow specific repair of cyclobutane pyrimidine dimers. To express the yeast DNA repair gene in human cells, the yeast PHR1 coding sequence was cloned into the mammalian expression vector pRSV4NEO-I. The resulting plasmid, pRSVPHR1, contains the coding sequence of the yeast gene, under control of transcription signals recognized by mammalian cells, and the dominant selectable gene neo. pRSVPHR1 was introduced into normal and XP SV40-transformed fibroblasts by the calcium phosphate coprecipitation technique, and G418-resistant clones were isolated. The level of PHR1 expression was determined by cytoplasmic RNA dot blots. Two clones, XP-3B and GM-20A, had high levels of expression.

  7. Moonlighting Proteins in Yeasts

    PubMed Central

    Gancedo, Carlos; Flores, Carmen-Lisset

    2008-01-01

    Proteins able to participate in unrelated biological processes have been grouped under the generic name of moonlighting proteins. Work with different yeast species has uncovered a great number of moonlighting proteins and shown their importance for adequate functioning of the yeast cell. Moonlighting activities in yeasts include such diverse functions as control of gene expression, organelle assembly, and modification of the activity of metabolic pathways. In this review, we consider several well-studied moonlighting proteins in different yeast species, paying attention to the experimental approaches used to identify them and the evidence that supports their participation in the unexpected function. Usually, moonlighting activities have been uncovered unexpectedly, and up to now, no satisfactory way to predict moonlighting activities has been found. Among the well-characterized moonlighting proteins in yeasts, enzymes from the glycolytic pathway appear to be prominent. For some cases, it is shown that despite close phylogenetic relationships, moonlighting activities are not necessarily conserved among yeast species. Organisms may utilize moonlighting to add a new layer of regulation to conventional regulatory networks. The existence of this type of proteins in yeasts should be taken into account when designing mutant screens or in attempts to model or modify yeast metabolism. PMID:18322039

  8. Yeast omnipotent supressor SUP1 (SUP45): nucleotide sequence of the wildtype and a mutant gene.

    PubMed

    Breining, P; Piepersberg, W

    1986-07-11

    The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined. The gene codes for a protein of 49 kD. The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part. The codon usage differs significantly from that of yeast ribosomal protein genes. However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S. cerevisiae has been found. A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria. The hypothetical function of this protein in control of translational fidelity is discussed.

  9. Expression of the yeast cpd1 gene in tobacco confers resistance to the fungal toxin cercosporin.

    PubMed

    Panagiotis, Madesis; Kritonas, Kalantidis; Irini, Nianiou Obeidat; Kiriaki, Chatzidimitriou; Nicolaos, Panopoulos; Athanasios, Tsaftaris

    2007-06-01

    Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.

  10. Identification and characterization of a gene and protein required for glycosylation in the yeast Golgi.

    PubMed

    Devlin, C; Ballou, C E

    1990-11-01

    The MNN2 gene of Saccharomyces cerevisiae has been cloned by complementation of the mnn2 mutant phenotype scored by a change in cell surface carbohydrate structure resulting from a lack of alpha 1----2-mannose branching in the outer chain. The gene was subcloned as a 3 kb DNA fragment that integrated at the MNN2 locus, and a gene disruption yielded the mnn2 phenotype. A lacZ-MNN2 gene fusion protein, produced in Escherichia coli, was used to raise a specific antiserum that recognized a 65 kD wild-type yeast protein. This MNN2 gene product lacks N-linked carbohydrate but appears to be an integral membrane protein. Overproduction of MNN2p does not enhance the alpha 1----2-mannosyltransferase activity of yeast cells. The results suggest that MNN2p is a Golgi-associated protein that is involved in mannoprotein sorting rather than glycosylation.

  11. An IF-FISH Approach for Covisualization of Gene Loci and Nuclear Architecture in Fission Yeast.

    PubMed

    Kim, K-D; Iwasaki, O; Noma, K

    2016-01-01

    Recent genomic studies have revealed that chromosomal structures are formed by a hierarchy of organizing processes ranging from gene associations, including interactions among enhancers and promoters, to topologically associating domain formations. Gene associations identified by these studies can be characterized by microscopic analyses. Fission yeast is a model organism, in which gene associations have been broadly mapped across the genome, although many of those associations have not been further examined by cell biological approaches. To address the technically challenging process of the visualization of associating gene loci in the fission yeast nuclei, we provide, in detail, an IF-FISH procedure that allows for covisualizing both gene loci and nuclear structural markers such as the nuclear membrane and nucleolus.

  12. Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

    PubMed

    Rautio, Jari J; Huuskonen, Anne; Vuokko, Heikki; Vidgren, Virve; Londesborough, John

    2007-09-01

    Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process. We monitored, using the transcript analysis with aid of affinity capture (TRAC) method, the expression of some 70 selected genes relevant to wort fermentation at high frequency through 9-10 day fermentations of very high gravity wort (25 degrees P) by an industrial lager strain. Rapid changes in expression occurred during the first hours of fermentations for several genes, e.g. genes involved in maltose metabolism, glycolysis and ergosterol synthesis were strongly upregulated 2-6 h after pitching. By the time yeast growth had stopped (72 h) and total sugars had dropped by about 50%, most selected genes had passed their highest expression levels and total mRNA was less than half the levels during growth. There was an unexpected upregulation of some genes of oxygen-requiring pathways during the final fermentation stages. For five genes, expression of both the Saccharomyces cerevisiae and S. bayanus components of the hybrid lager strain were determined. Expression profiles were either markedly different (ADH1, ERG3) or very similar (MALx1, ILV5, ATF1) between these two components. By frequent analysis of a chosen set of genes, TRAC provided a detailed and dynamic picture of the physiological state of the fermenting yeast. This approach offers a possible way to monitor and optimize the performance of yeast in a complex process environment.

  13. Dynamics and modeling of temperature-regulated gene product expression in recombinant yeast fermentation.

    PubMed

    Cheng, C; Yang, S T

    1996-06-20

    The dynamic response of temperature-regulated gene expression in the recombinant yeast Saccharomyces cerevisiae, strain XK1-C2 carrying plasmid pSXR125, to temperature changes during fed-batch and continuous (chemostat) cultures was studied. The production of the gene product, beta-galactosidase, in the yeast cell is sensitive to the growth temperature. Gene expression of this product was fully turned on or off by temperature shifts between 24 and 30 degrees C. However, the response for gene turn-on and turn-off in this recombinant yeast was slow, requiring from several hours to over 10 h to fully appear. The continuous reactor took 30-60 h after the temperature shift to reach a new steady state. A dynamic process model was developed to simulate the reactor and cell responses to temperature shift. A first-order model was used to account for the effect of dilution rate on the change of protein concentration in the chemostat. It was found that cell response in gene expression to temperature shift followed first-order plus dead-time dynamics. Also, the response time for gene expression to temperature shift varied with specific growth rate or dilution rate of the continuous reactor. In general, the response was slower at a higher dilution rate and for gene turn-on than for gene turn-off.

  14. Pervasive and Persistent Redundancy among Duplicated Genes in Yeast

    PubMed Central

    Dean, E. Jedediah; Davis, Jerel C.; Davis, Ronald W.; Petrov, Dmitri A.

    2008-01-01

    The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (∼100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time. PMID:18604285

  15. Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109.

    PubMed

    Wang, Zhi-Peng; Xu, Hong-Mei; Wang, Guang-Yuan; Chi, Zhe; Chi, Zhen-Ming

    2013-04-01

    In this study, the MIG1 gene in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109 (the parent yeast) was disrupted and the disruptant M25 obtained could grow in yeast nitrogen base-N5000 medium without uracil or the medium with 2-deoxy-D-glucose. It was found that the cells of the disruptant M25 had more lipid bodies than those of its parent yeast. The disruptant M25 contained 48.7% (w/w) of oil based on its cell weight while the parent yeast only contained 36.0% (w/w) of oil. Transcript levels of many genes relevant to lipid biosynthesis in the disruptant M25 were enhanced compared to those of the same genes in the parent yeast. However, transcript level of the MFE1 gene, one of the genes relevant to fatty acid degradation was reduced in the disruptant M25 compared to that of the same gene in the parent yeast. Such changes in gene expression profile may cause the increased lipid biosynthesis in the disruptant M25. Biosynthesis of C18:1 fatty acid in the disruptant M25 was greatly enhanced compared to that in the parent yeast. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Yeast prion architecture explains how proteins can be genes

    NASA Astrophysics Data System (ADS)

    Wickner, Reed

    2013-03-01

    Prions (infectious proteins) transmit information without an accompanying DNA or RNA. Most yeast prions are self-propagating amyloids that inactivate a normally functional protein. A single protein can become any of several prion variants, with different manifestations due to different amyloid structures. We showed that the yeast prion amyloids of Ure2p, Sup35p and Rnq1p are folded in-register parallel beta sheets using solid state NMR dipolar recoupling experiments, mass-per-filament-length measurements, and filament diameter measurements. The extent of beta sheet structure, measured by chemical shifts in solid-state NMR and acquired protease-resistance on amyloid formation, combined with the measured filament diameters, imply that the beta sheets must be folded along the long axis of the filament. We speculate that prion variants of a single protein sequence differ in the location of these folds. Favorable interactions between identical side chains must hold these structures in-register. The same interactions must guide an unstructured monomer joining the end of a filament to assume the same conformation as molecules already in the filament, with the turns at the same locations. In this way, a protein can template its own conformation, in analogy to the ability of a DNA molecule to template its sequence by specific base-pairing. Bldg. 8, Room 225, NIH, 8 Center Drive MSC 0830, Bethesda, MD 20892-0830, wickner@helix.nih.gov, 301-496-3452

  17. Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts.

    PubMed

    Deregowska, Anna; Skoneczny, Marek; Adamczyk, Jagoda; Kwiatkowska, Aleksandra; Rawska, Ewa; Skoneczna, Adrianna; Lewinska, Anna; Wnuk, Maciej

    2015-10-13

    Industrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains.

  18. Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts

    PubMed Central

    Adamczyk, Jagoda; Kwiatkowska, Aleksandra; Rawska, Ewa; Skoneczna, Adrianna

    2015-01-01

    Industrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains. PMID:26384347

  19. Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

    PubMed

    Kim, Yu-Young; Kim, Do-Young; Shim, Donghwan; Song, Won-Yong; Lee, Joohyun; Schroeder, Julian I; Kim, Sanguk; Moran, Nava; Lee, Youngsook

    2008-06-06

    Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

  20. Using a Euclid distance discriminant method to find protein coding genes in the yeast genome.

    PubMed

    Zhang, Chun-Ting; Wang, Ju; Zhang, Ren

    2002-02-01

    The Euclid distance discriminant method is used to find protein coding genes in the yeast genome, based on the single nucleotide frequencies at three codon positions in the ORFs. The method is extremely simple and may be extended to find genes in prokaryotic genomes or eukaryotic genomes with less introns. Six-fold cross-validation tests have demonstrated that the accuracy of the algorithm is better than 93%. Based on this, it is found that the total number of protein coding genes in the yeast genome is less than or equal to 5579 only, about 3.8-7.0% less than 5800-6000, which is currently widely accepted. The base compositions at three codon positions are analyzed in details using a graphic method. The result shows that the preference codons adopted by yeast genes are of the RGW type, where R, G and W indicate the bases of purine, non-G and A/T, whereas the 'codons' in the intergenic sequences are of the form NNN, where N denotes any base. This fact constitutes the basis of the algorithm to distinguish between coding and non-coding ORFs in the yeast genome. The names of putative non-coding ORFs are listed here in detail.

  1. The Yeast HAL1 Gene Improves Salt Tolerance of Transgenic Tomato1

    PubMed Central

    Gisbert, Carmina; Rus, Ana M.; Bolarín, M. Carmen; López-Coronado, J. Miguel; Arrillaga, Isabel; Montesinos, Consuelo; Caro, Manuel; Serrano, Ramon; Moreno, Vicente

    2000-01-01

    Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K+ concentration and decreasing intracellular Na+ during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K+ to Na+ ratios showed that transgenic lines were able to retain more K+ than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast. PMID:10806256

  2. High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

    USDA-ARS?s Scientific Manuscript database

    To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled...

  3. Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

    PubMed

    Hayashi, Kazuhiro; Ogiyama, Yuki; Yokomi, Kazumasa; Nakagawa, Tsuyoshi; Kaino, Tomohiro; Kawamukai, Makoto

    2014-01-01

    Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

  4. Evidence for ORC-dependent repression of budding yeast genes induced by starvation and other stresses.

    PubMed

    Ramachandran, Lakshmi; Burhans, Debra T; Laun, Peter; Wang, Jianxin; Liang, Ping; Weinberger, Martin; Wissing, Silke; Jarolim, Stefanie; Suter, Bernhard; Madeo, Frank; Breitenbach, Michael; Burhans, William C

    2006-08-01

    The highly conserved origin recognition complex (ORC) is required for repressing genes in the silent mating type loci of budding yeast. Here we report that at a non-permissive temperature, the temperature-sensitive orc2-1 mutation induces the expression of more than 500 genes, the majority of which are also induced during starvation of wild-type cells. Many genes induced by starvation or by the orc2-1 mutation are also induced by inactivation of proteins required for chromatin-mediated repression of transcription. Genes induced by the orc2-1 mutation, starvation, or inactivation of repressor proteins, map near ORC-binding loci significantly more frequently compared to all genes. Genes repressed by starvation map near ORC-binding sites less frequently compared to all genes, which suggests they have been evolutionarily excluded from regions of repressive chromatin near ORC-binding sites. Deletion of sequences containing ORC-binding sites near the DAL2 and DAL4 genes in the DAL gene cluster, which are induced by either the orc2-1 mutation or by starvation, constitutively activates these genes and abolishes their activation by the orc2-1 mutation. Our findings suggest a role for ORC in the repression of a large number of budding yeast genes induced by starvation or other aspects of a deleterious environment.

  5. sigma, a repetitive element found adjacent to tRNA genes of yeast.

    PubMed Central

    del Rey, F J; Donahue, T F; Fink, G R

    1982-01-01

    sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome. The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats. The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence. sigma is located 16 or 18 bp from the 5' end of several tRNA genes. Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains. Images PMID:6287468

  6. Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces dendrorhous.

    PubMed

    Córdova, Pamela; Gonzalez, Ana-María; Nelson, David R; Gutiérrez, María-Soledad; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2017-07-19

    The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. The carotenogenic yeast X. dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the

  7. A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica.

    PubMed

    Neuvéglise, C; Nicauda, J M; Ross-Macdonald, P; Gaillardin, C

    1998-06-15

    A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.

  8. Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

    PubMed Central

    Chen, X. J.; Clark-Walker, G. D.

    1993-01-01

    Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes. PMID:8454202

  9. The use of yeast genetic diversity for agricultural and biotechnological applications

    USDA-ARS?s Scientific Manuscript database

    Many yeasts have been shown to be effective for biocontrol of field, greenhouse, and storage diseases of agricultural crops. Yeasts are generally regarded as safe for a wide variety of applications and some species establish large populations on leaf and fruit surfaces, resulting in disease control ...

  10. The use of yeast genetic diversity for agricultural and biotechnological applications

    USDA-ARS?s Scientific Manuscript database

    Many types of yeast have been shown to be effective for biocontrol of field, greenhouse, and storage diseases of agricultural crops. Yeasts are generally regarded as safe for a wide variety of applications and some species establish large populations on leaf and fruit surfaces, resulting in disease ...

  11. Analysis of gene expression profiles of Lactobacillus paracasei induced by direct contact with Saccharomyces cerevisiae through recognition of yeast mannan

    PubMed Central

    YAMASAKI-YASHIKI, Shino; SAWADA, Hiroshi; KINO-OKA, Masahiro; KATAKURA, Yoshio

    2016-01-01

    Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of Lactobacillus paracasei ATCC 334 co-cultured with Saccharomyces cerevisiae IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast. PMID:28243547

  12. Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica.

    PubMed

    Mirończuk, Aleksandra M; Biegalska, Anna; Dobrowolski, Adam

    2017-01-01

    Erythritol, a four-carbon polyol synthesized by microorganisms as an osmoprotectant, is a natural sweetener produced on an industrial scale for decades. Despite the fact that the yeast Yarrowia lipolytica has been reported since the 1970s as an erythritol producer, the metabolic pathway of this polyol has never been characterized. It was shown that erythritol synthesis in yeast occurs via the pentose phosphate pathway (PPP). The oleaginous yeast Y. lipolytica is a good host for converting inexpensive glycerol into a value-added product such as erythritol. Glycerol is a renewable feedstock which is produced on a large scale as a waste product by many branches of industry. In this study, we functionally overexpressed four genes involved in the pentose phosphate pathway (PPP): gene YALI0E06479g encoding transketolase (TKL1), gene YALI0F15587g encoding transaldolase (TAL1), gene YALI0E22649g encoding glucose-6-phosphate dehydrogenase (ZWF1), and gene YALI0B15598g encoding 6-phosphogluconate dehydrogenase (GND1). Here, we show that the crucial gene for erythritol synthesis in Y. lipolytica is transketolase. Overexpression of this gene results in a twofold improvement in erythritol synthesis during a shake-flask experiment (58 g/L). Moreover, overexpression of TKL1 allows for efficient production of erythritol independently from the supplied dissolved oxygen. Fermentation conducted in a 5-L bioreactor at low agitation results in almost 70% higher titer of erythritol over the control strain. This work presents the importance of the PPP in erythritol synthesis and the feasibility for economic production of erythritol from glycerol by the yeast Y. lipolytica.

  13. Phylogeny of tremellomycetous yeasts and related dimorphic and filamentous basidiomycetes reconstructed from multiple gene sequence analyses

    PubMed Central

    Liu, X.-Z.; Wang, Q.-M.; Theelen, B.; Groenewald, M.; Bai, F.-Y.; Boekhout, T.

    2015-01-01

    The Tremellomycetes (Basidiomycota) contains a large number of unicellular and dimorphic fungi with stable free-living unicellular states in their life cycles. These fungi have been conventionally classified as basidiomycetous yeasts based on physiological and biochemical characteristics. Many currently recognised genera of these yeasts are mainly defined based on phenotypical characters and are highly polyphyletic. Here we reconstructed the phylogeny of the majority of described anamorphic and teleomorphic tremellomycetous yeasts using Bayesian inference, maximum likelihood, and neighbour-joining analyses based on the sequences of seven genes, including three rRNA genes, namely the small subunit of the ribosomal DNA (rDNA), D1/D2 domains of the large subunit rDNA, and the internal transcribed spacer regions (ITS 1 and 2) of rDNA including 5.8S rDNA; and four protein-coding genes, namely the two subunits of the RNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB). With the consideration of morphological, physiological and chemotaxonomic characters and the congruence of phylogenies inferred from analyses using different algorithms based on different data sets consisting of the combined seven genes, the three rRNA genes, and the individual protein-coding genes, five major lineages corresponding to the orders Cystofilobasidiales, Filobasidiales, Holtermanniales, Tremellales, and Trichosporonales were resolved. A total of 45 strongly supported monophyletic clades with multiple species and 23 single species clades were recognised. This phylogenetic framework will be the basis for the proposal of an updated taxonomic system of tremellomycetous yeasts that will be compatible with the current taxonomic system of filamentous basidiomycetes accommodating the ‘one fungus, one name’ principle. PMID:26955196

  14. Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

    PubMed Central

    Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

    2016-01-01

    Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

  15. Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

    PubMed

    Jin, Ke; Musso, Gabriel; Vlasblom, James; Jessulat, Matthew; Deineko, Viktor; Negroni, Jacopo; Mosca, Roberto; Malty, Ramy; Nguyen-Tran, Diem-Hang; Aoki, Hiroyuki; Minic, Zoran; Freywald, Tanya; Phanse, Sadhna; Xiang, Qian; Freywald, Andrew; Aloy, Patrick; Zhang, Zhaolei; Babu, Mohan

    2015-02-06

    Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

  16. Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

    PubMed

    Narcross, Lauren; Bourgeois, Leanne; Fossati, Elena; Burton, Euan; Martin, Vincent J J

    2016-12-16

    The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

  17. Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains.

    PubMed

    Rivero, Damaríz; Berná, Luisa; Stefanini, Irene; Baruffini, Enrico; Bergerat, Agnes; Csikász-Nagy, Attila; De Filippo, Carlotta; Cavalieri, Duccio

    2015-08-01

    The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p-dependent altruistic and contact-dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast-growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.

  18. A general lack of compensation for gene dosage in yeast

    PubMed Central

    Springer, Michael; Weissman, Jonathan S; Kirschner, Marc W

    2010-01-01

    Gene copy number variation has been discovered in humans, between related species, and in different cancer tissues, but it is unclear how much of this genomic-level variation leads to changes in the level of protein abundance. To address this, we eliminated one of the two genomic copies of 730 different genes in Saccharomyces cerevisiae and asked how often a 50% reduction in gene dosage leads to a 50% reduction in protein level. For at least 80% of genes tested, and under several environmental conditions, it does: protein levels in the heterozygous strain are close to 50% of wild type. For <5% of the genes tested, the protein levels in the heterozygote are maintained at nearly wild-type levels. These experiments show that protein levels are not, in general, directly monitored and adjusted to a desired level. Combined with fitness data, this implies that proteins are expressed at levels higher than necessary for survival. PMID:20461075

  19. Using Multi-Instance Hierarchical Clustering Learning System to Predict Yeast Gene Function

    PubMed Central

    Liao, Bo; Li, Yun; Jiang, Yan; Cai, Lijun

    2014-01-01

    Time-course gene expression datasets, which record continuous biological processes of genes, have recently been used to predict gene function. However, only few positive genes can be obtained from annotation databases, such as gene ontology (GO). To obtain more useful information and effectively predict gene function, gene annotations are clustered together to form a learnable and effective learning system. In this paper, we propose a novel multi-instance hierarchical clustering (MIHC) method to establish a learning system by clustering GO and compare this method with other learning system establishment methods. Multi-label support vector machine classifier and multi-label K-nearest neighbor classifier are used to verify these methods in four yeast time-course gene expression datasets. The MIHC method shows good performance, which serves as a guide to annotators or refines the annotation in detail. PMID:24621610

  20. The HLA genes and their diverse polymorphism.

    PubMed

    Mehra, N K

    2000-08-01

    Advanced DNA level studies based on HLA class II sequence analysis have revealed considerable diversity in HLA among Asian Indians. High resolution typing of specific alleles such as DR2 and DR4 in the HLA class II region by PCR-SSP or SSOP hybridization and their associated DR-DQ haplotypes have helped to detect unique haplotypes and novel alleles which have subsequently been confirmed by sequencing. Incidentally, remarkable stability has been maintained in several other DRB1 alleles viz. DR1, DR7, DR9 and DR10. The ARMS-PCR technology has been found to be particularly useful for typing HLA-A, HLA-B and HLA-Cw alleles. These technologies are far superior over serological methods. Our studies have shown remarkable heterogeneity of common HLA-A and B alleles in Asian Indians. Molecular subtyping of HLA-A2 revealed that subtype A(*)0211 is found only in Indian population and may be the result of selection pressure in this population. Investigations into polymorphism in the HLA-B27 gene revealed that subtypes common both to the western caucasians and orientals occur in the Indian population. It is apparent that the population of the Indian subcontinent, placed as it is between the Caucasoids and Negroids on one hand and Australoids and Mongoloids on the other, provides a rich source of many HLA haplotypes. While the most frequent Caucasian haplotypes occur with a reasonable frequency in Asian Indians, those found predominantly in other ethnic groups (e.g., australian Aborigines and populations of Oceania, China and Japan) are also detected. Knowledge on this is most important for donor selection during organ and bone marrow transplantation and for designing MHC targeted vaccines in specific diseases.

  1. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    PubMed

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  2. New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast.

    PubMed

    Wosika, Victoria; Durandau, Eric; Varidel, Clémence; Aymoz, Delphine; Schmitt, Marta; Pelet, Serge

    2016-12-01

    The tractability of the budding yeast genome has provided many insights into the fundamental mechanisms regulating cellular life. With the advent of synthetic biology and single-cell measurements, novel tools are required to manipulate the yeast genome in a more controlled manner. We present, here, a new family of yeast shuttle vectors called single integration vectors (pSIV). Upon transformation in yeast, these plasmids replace the entire deficient auxotrophy marker locus by a cassette containing an exogenous marker. As shown using flow cytometry, this complete replacement results in a unique integration of the desired DNA fragment at the marker locus. In addition, a second transcriptional unit can be inserted to achieve the simultaneous integration of two constructs. The selection marker cassettes, present in the pSIV, were also used to generate a complete set of gene tagging plasmids (pGT) encompassing a large palette of fluorescent proteins, from a cyan fluorescent protein to a near-infrared tandem dimer red fluorescent protein. These tagging cassettes are orthogonal to each other thanks to the use of different TEF promoter and terminator couples, thereby avoiding marker cassette switching and favoring integration in the desired locus. In summary, we have created two sets of robust molecular tools for the precise genetic manipulation of the budding yeast.

  3. Stable chromosomal integration of the entire nitrogen fixation gene cluster from Klebsiella pneumoniae in yeast.

    PubMed Central

    Zamir, A; Maina, C V; Fink, G R; Szalay, A A

    1981-01-01

    A bacterial plasmid containing the entire nitrogen fixation (nif) gene cluster (consisting of at least 15 genes) from Klebsiella pneumoniae was used in conjunction with an Escherichia coli-yeast shuttle plasmid containing the yeast his4 gene cluster to cotransform a his4- recipient strain of Saccharomyces cerevisiae. Of 87 histidine-independent clones screened, 2 contained nif DNA. Restriction and hybridization analyses showed that two copies of the nif plasmid (46 kilobases each) are integrated in tandem in the recipient chromosome by recombination between homologous regions in the transforming plasmids. Chromosomal integration was also verified by tetrad analysis, showing that the nif DNA behaved in meiosis like a Mendelian element. During mitotic growth, one of the two copies of the nif region is frequently lost. The remaining copy of nif is stable, even after 40 generations in nonselective medium. Images PMID:6267596

  4. Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

    PubMed Central

    Převorovský, Martin; Oravcová, Martina; Tvarůžková, Jarmila; Zach, Róbert; Folk, Petr; Půta, František; Bähler, Jürg

    2015-01-01

    Background Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. Methodology/Principal Findings Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. Conclusions/Significance Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast. PMID:26366556

  5. Yeast model identifies ENTPD6 as a potential non-obstructive azoospermia pathogenic gene

    PubMed Central

    Wang, Qian; Liu, Chao; Tang, Chaoming; Guo, Huiping; Liu, Yujiao; Wang, Lina; Zhao, Haichao; Shang, Yongliang; Wen, Yang; Lin, Yuan; Zhou, Tao; Zhou, Zuomin; Dong, Wen; Hu, Zhibin; Guo, Xuejiang; Sha, Jiahao; Li, Wei

    2015-01-01

    Approximately ten percent of male infertility is caused by non-obstructive azoospermia (NOA), but the etiologies of many NOA remain elusive. Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity. However, those variants that lack genome-wide significance might still be essential for fertility. Functional analysis of genes surrounding these variants in Drosophila identified some spermatogenesis-essential genes. As a complementary method of Drosophila screening, SK1 background Saccharomvces cerevisiae was used as a model to screen meiosis-related genes from the NOA GWAS data in this study. After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene. The deletion of GDA1 resulted in the failure of yeast sporulation. Further investigations showed that Gda1p was important for pre-meiotic S phase entry. Interestingly, the meiotic role of Gda1p was dependent on its guanosine diphosphatase activity, but not it’s cytoplasmic, transmembrane or stem domains. These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA. PMID:26152596

  6. Yeast model identifies ENTPD6 as a potential non-obstructive azoospermia pathogenic gene.

    PubMed

    Wang, Qian; Liu, Chao; Tang, Chaoming; Guo, Huiping; Liu, Yujiao; Wang, Lina; Zhao, Haichao; Shang, Yongliang; Wen, Yang; Lin, Yuan; Zhou, Tao; Zhou, Zuomin; Dong, Wen; Hu, Zhibin; Guo, Xuejiang; Sha, Jiahao; Li, Wei

    2015-07-08

    Approximately ten percent of male infertility is caused by non-obstructive azoospermia (NOA), but the etiologies of many NOA remain elusive. Recently, a genome-wide association study (GWAS) of NOA in Han Chinese men was conducted, and only a few genetic variants associated with NOA were found, which might have resulted from genetic heterogeneity. However, those variants that lack genome-wide significance might still be essential for fertility. Functional analysis of genes surrounding these variants in Drosophila identified some spermatogenesis-essential genes. As a complementary method of Drosophila screening, SK1 background Saccharomvces cerevisiae was used as a model to screen meiosis-related genes from the NOA GWAS data in this study. After functional screening, GDA1 (orthologous to humanENTPD6) was found to be a novel meiosis-related gene. The deletion of GDA1 resulted in the failure of yeast sporulation. Further investigations showed that Gda1p was important for pre-meiotic S phase entry. Interestingly, the meiotic role of Gda1p was dependent on its guanosine diphosphatase activity, but not it's cytoplasmic, transmembrane or stem domains. These yeast data suggest that ENTPD6 may be a novel meiosis-associated NOA-related gene, and the yeast model provides a good approach to analyze GWAS results of NOA.

  7. Retrovirus-like vectors for Saccharomyces cerevisiae: integration of foreign genes controlled by efficient promoters into yeast chromosomal DNA.

    PubMed

    Jacobs, E; Dewerchin, M; Boeke, J D

    1988-07-30

    Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced. These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene. Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome. A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette. This may be because it does not allow the transcription run-through necessary for Ty1 transposition. Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.

  8. Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

    PubMed Central

    Banerjee, Shataparna; Khandelia, Piyush; Melangath, Geetha; Bashir, Samirul; Nagampalli, Vijaykrishna

    2013-01-01

    The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7+ (spslu7+) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3′ splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3′ splice site distance, intron length, and the impact of its A/U content at the 5′ end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1+, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly. PMID:23754748

  9. Microbial Terroir in Chilean Valleys: Diversity of Non-conventional Yeast

    PubMed Central

    Jara, Carla; Laurie, V. Felipe; Mas, Albert; Romero, Jaime

    2016-01-01

    In this study, the presence of non-conventional yeast associated with vineyards located between latitudes 30°S and 36°S was examined, including the valleys of Limarí, Casablanca, Maipo, Colchagua, Maule, and Itata. The microbial fingerprinting in each valley was examined based on the specific quantification of yeast of enological interest. Grape–berries were sampled to evaluate the presence and load of non-conventional yeast with enological potential, such as Metschnikowia, Hanseniaspora, Torulaspora, Debaryomyces, Meyerozyma, and Rhodotorula. These yeasts were present in all vineyards studied but with varying loads depending on the valley sampled. No identical fingerprints were observed; however, similarities and differences could be observed among the microbial profiles of each valley. A co-variation in the loads of Metschnikowia and Hanseniaspora with latitude was observed, showing high loads in the Casablanca and Itata valleys, which was coincident with the higher relative humidity or rainfall of those areas. Non-conventional yeasts were also isolated and identified after sequencing molecular markers. Potentially good aromatic properties were also screened among the isolates, resulting in the selection of mostly Metschnikowia and Hanseniaspora isolates. Finally, our results suggest that microbial terroir might be affected by climatic conditions such as relative humidity and rainfall, especially impacting the load of non-conventional yeast. In this study, the microbial fingerprint for yeast in Chilean vineyards is reported for the first time revealing an opportunity to study the contribution of this assembly of microorganisms to the final product. PMID:27242693

  10. Microbial Terroir in Chilean Valleys: Diversity of Non-conventional Yeast.

    PubMed

    Jara, Carla; Laurie, V Felipe; Mas, Albert; Romero, Jaime

    2016-01-01

    In this study, the presence of non-conventional yeast associated with vineyards located between latitudes 30°S and 36°S was examined, including the valleys of Limarí, Casablanca, Maipo, Colchagua, Maule, and Itata. The microbial fingerprinting in each valley was examined based on the specific quantification of yeast of enological interest. Grape-berries were sampled to evaluate the presence and load of non-conventional yeast with enological potential, such as Metschnikowia, Hanseniaspora, Torulaspora, Debaryomyces, Meyerozyma, and Rhodotorula. These yeasts were present in all vineyards studied but with varying loads depending on the valley sampled. No identical fingerprints were observed; however, similarities and differences could be observed among the microbial profiles of each valley. A co-variation in the loads of Metschnikowia and Hanseniaspora with latitude was observed, showing high loads in the Casablanca and Itata valleys, which was coincident with the higher relative humidity or rainfall of those areas. Non-conventional yeasts were also isolated and identified after sequencing molecular markers. Potentially good aromatic properties were also screened among the isolates, resulting in the selection of mostly Metschnikowia and Hanseniaspora isolates. Finally, our results suggest that microbial terroir might be affected by climatic conditions such as relative humidity and rainfall, especially impacting the load of non-conventional yeast. In this study, the microbial fingerprint for yeast in Chilean vineyards is reported for the first time revealing an opportunity to study the contribution of this assembly of microorganisms to the final product.

  11. Diversity of Carotenoid Synthesis Gene Clusters from Environmental Enterobacteriaceae Strains

    PubMed Central

    Sedkova, Natalia; Tao, Luan; Rouvière, Pierre E.; Cheng, Qiong

    2005-01-01

    Eight Enterobacteriaceae strains that produce zeaxanthin and derivatives of this compound were isolated from a variety of environmental samples. Phylogenetic analysis showed that these strains grouped with different clusters of Erwinia type strains. Four strains representing the phylogenetic diversity were chosen for further characterization, which revealed their genetic diversity as well as their biochemical diversity. The carotenoid synthesis gene clusters cloned from the four strains had three different gene organizations. Two of the gene clusters, those from strains DC416 and DC260, had the classical organization crtEXYIBZ; the gene cluster from DC413 had the rare organization crtE-idi-XYIBZ; and the gene cluster from DC404 had the unique organization crtE-idi-YIBZ. Besides the diversity in genetic organization, these genes also exhibited considerable sequence diversity. On average, they exhibited 60 to 70% identity with each other, as well as with the corresponding genes of the Pantoea type strains. The four different clusters were individually expressed in Escherichia coli, and the two idi-containing clusters gave more than fivefold-higher carotenoid titers than the two clusters lacking idi. Expression of the crtEYIB genes with and without idi confirmed the effect of increasing carotenoid titer by the type II idi gene linked with the carotenoid synthesis gene clusters. PMID:16332796

  12. Changes in expression of oxidative stress related genes in grapefruit peel in response to yeast Metschnikowia fructicola

    USDA-ARS?s Scientific Manuscript database

    To gain insight into the mode of action of the yeast biocontrol agent, Metschnikowia fructicola, the transcription profiles of genes involved in oxidative stress were studied in grapefruit (Citrus paradis, 'Star Ruby') surface wounds following the application of the yeast antagonist. Three transcri...

  13. Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies.

    PubMed

    van Helden, J; André, B; Collado-Vides, J

    1998-09-04

    We present here a simple and fast method allowing the isolation of DNA binding sites for transcription factors from families of coregulated genes, with results illustrated in Saccharomyces cerevisiae. Although conceptually simple, the algorithm proved efficient for extracting, from most of the yeast regulatory families analyzed, the upstream regulatory sequences which had been previously found by experimental analysis. Furthermore, putative new regulatory sites are predicted within upstream regions of several regulons. The method is based on the detection of over-represented oligonucleotides. A specificity of this approach is to define the statistical significance of a site based on tables of oligonucleotide frequencies observed in all non-coding sequences from the yeast genome. In contrast with heuristic methods, this oligonucleotide analysis is rigorous and exhaustive. Its range of detection is however limited to relatively simple patterns: short motifs with a highly conserved core. These features seem to be shared by a good number of regulatory sites in yeast. This, and similar methods, should be increasingly required to identify unknown regulatory elements within the numerous new coregulated families resulting from measurements of gene expression levels at the genomic scale. All tools described here are available on the web at the site http://copan.cifn.unam.mx/Computational_Biology/ yeast-tools Copyright 1998 Academic Press

  14. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    PubMed Central

    Chaillot, Julien; Cook, Michael A.; Corbeil, Jacques; Sellam, Adnane

    2016-01-01

    One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. PMID:28040776

  15. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans.

    PubMed

    Chaillot, Julien; Cook, Michael A; Corbeil, Jacques; Sellam, Adnane

    2017-02-09

    One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. Copyright © 2017 Chaillot et al.

  16. Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress.

    PubMed

    James, Tharappel C; Usher, Jane; Campbell, Susan; Bond, Ursula

    2008-03-01

    A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress.

  17. Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

    PubMed Central

    Ha, S B; Smith, A P; Howden, R; Dietrich, W M; Bugg, S; O'Connell, M J; Goldsbrough, P B; Cobbett, C S

    1999-01-01

    Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species. PMID:10368185

  18. Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast

    PubMed Central

    Rodriguez-Vargas, Sonia; Estruch, Francisco; Randez-Gil, Francisca

    2002-01-01

    We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast. PMID:12039763

  19. Diversity of culturable yeasts in phylloplane of sugarcane in Thailand and their capability to produce indole-3-acetic acid.

    PubMed

    Limtong, Savitree; Kaewwichian, Rungluk; Yongmanitchai, Wichien; Kawasaki, Hiroko

    2014-06-01

    Yeasts were isolated by the enrichment technique from the phylloplane of 94 samples of sugarcane leaf collected from seven provinces in Thailand. All sugarcane leaf samples contained yeasts and 158 yeast strains were obtained. On the basis of the D1/D2 domain of the large subunit rRNA gene sequence analysis, 144 strains were identified to 24 known species in 14 genera belonging to the Ascomycota viz. Candida akabanensis, Candida dendronema, Candida mesorugosa, Candida michaelii, Candida nivariensis, Candida rugosa, Candida orthopsilosis, Candida quercitrusa, Candida tropicalis, Candida xylopsoci, Cyberlindnera fabianii, Cyberlindnera rhodanensis, Debaryomyces nepalensis, Hannaella aff. coprosmaensis, Hanseniaspora guilliermondii, Kluyveromyces marxianus, Lachancea thermotolerans, Lodderomyces elongisporus, Metschnikowia koreensis, Meyerozyma caribbica, Millerozyma koratensis, Pichia kudriavzevii, Torulaspora delbrueckii and Wickerhamomyces edaphicus, and 12 species in six genera of the Basidiomycota viz . Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus rajasthanensis, Kwoniella heveanensis, Rhodosporidium fluviale, Rhodosporidium paludigenum, Rhodotorula mucilaginosa, Rhodotorula sesimbrana, Rhodotorula taiwanensis, Sporidiobolus ruineniae, Sporobolomyces carnicolor and Sporobolomyces nylandii. Seven strains were identical or similar to four undescribed species. Another seven strains represented four novels species in the genus Metschnikowia, Nakazawaea, Wickerhamomyces and Yamadazyma. The results revealed 69 % of the isolated strains were ascomycete yeasts and 31 % were basidiomycete yeast. The most prevalent species was M. caribbica with a 23 % frequency of occurrence followed by Rh. taiwanensis (11 %) and C. tropicalis (10 %). All strains were assessed for indole-3-acetic acid (IAA) producing capability showing that 69 strains had the capability of producing IAA when cultivated in yeast extract peptone dextrose broth supplemented with 1

  20. Measure of synonymous codon usage diversity among genes in bacteria.

    PubMed

    Suzuki, Haruo; Saito, Rintaro; Tomita, Masaru

    2009-06-01

    In many bacteria, intragenomic diversity in synonymous codon usage among genes has been reported. However, no quantitative attempt has been made to compare the diversity levels among different genomes. Here, we introduce a mean dissimilarity-based index (Dmean) for quantifying the level of diversity in synonymous codon usage among all genes within a genome. The application of Dmean to 268 bacterial genomes shows that in bacteria with extremely biased genomic G+C compositions there is little diversity in synonymous codon usage among genes. Furthermore, our findings contradict previous reports. For example, a low level of diversity in codon usage among genes has been reported for Helicobacter pylori, but based on Dmean, the diversity level of this species is higher than those of more than half of bacteria tested here. The discrepancies between our findings and previous reports are probably due to differences in the methods used for measuring codon usage diversity. We recommend that Dmean be used to measure the diversity level of codon usage among genes. This measure can be applied to other compositional features such as amino acid usage and dinucleotide relative abundance as a genomic signature.

  1. SUMO functions in constitutive transcription and during activation of inducible genes in yeast.

    PubMed

    Rosonina, Emanuel; Duncan, Sarah M; Manley, James L

    2010-06-15

    Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

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

    SciTech Connect

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-02-07

    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.

  3. Comparison of the MTT1- and MAL31-like maltose transporter genes in lager yeast strains.

    PubMed

    Dietvorst, Judith; Walsh, Mike C; van Heusden, G Paul H; Steensma, H Yde

    2010-09-01

    Maltose transporter genes were isolated from four lager yeast strains and sequenced. All four strains contain at least two different types of maltose transporter genes, MTT1 and MAL31. In addition, 'long' 2.7 kb, and 'short' 2.4 kb, versions of each type exist. The size difference is caused by the insertion of two repeats of 147 bp into the promoter regions of the long versions of the genes. As a consequence of the insertion, two Mal63-binding sites move 294 bp away from the transcription initiation site. The 2.4- and 2.7-kb versions are further highly similar. Only the 2.4-kb versions and not the 2.7-kb versions of MTT1 could restore the rapid growth of lager yeast strain A15 on maltotriose in the presence of antimycin A. These results suggest that insertion of the two repeats into the promoter region of the 'long versions' of MTT1 genes led to a diminished expression of these genes. None of the tested long and short versions of the MAL31 genes were able to restore this growth. As the promoter regions of the MTT1 and MAL31 genes are identical, small differences in the protein sequence may be responsible for the different properties of these genes.

  4. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    SciTech Connect

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  5. Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota).

    PubMed

    Teixeira, M M; Moreno, L F; Stielow, B J; Muszewska, A; Hainaut, M; Gonzaga, L; Abouelleil, A; Patané, J S L; Priest, M; Souza, R; Young, S; Ferreira, K S; Zeng, Q; da Cunha, M M L; Gladki, A; Barker, B; Vicente, V A; de Souza, E M; Almeida, S; Henrissat, B; Vasconcelos, A T R; Deng, S; Voglmayr, H; Moussa, T A A; Gorbushina, A; Felipe, M S S; Cuomo, C A; de Hoog, G Sybren

    2017-03-01

    The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and

  6. An essential yeast gene encoding a TTAGGG repeat-binding protein.

    PubMed Central

    Brigati, C; Kurtz, S; Balderes, D; Vidali, G; Shore, D

    1993-01-01

    A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results. Images PMID:8423796

  7. Functional analysis to identify genes in wine yeast adaptation to low-temperature fermentation.

    PubMed

    Salvadó, Z; Chiva, R; Rozès, N; Cordero-Otero, R; Guillamón, J M

    2012-07-01

      To identify genes and proteins involved in adaptation to low-temperature fermentations in a commercial wine yeast.   Nine proteins were identified as representing the most significant changes in proteomic maps during the first 24 h of fermentation at low (13°C) and standard temperature (25°C). These proteins were mainly involved in stress response and in glucose and nitrogen metabolism. Transcription analysis of the genes encoding most of these proteins within the same time frame of wine fermentation presented a good correlation with proteomic data. Knockout and overexpressing strains of some of these genes were constructed and tested to evaluate their ability to start the fermentation process. The strain overexpressing ILV5 improved its fermentation activity in the first hours of fermentation. This strain showed a quicker process of mitochondrial degeneration, an altered intracellular amino acid profile and laxer nitrogen catabolite repression regulation.   The proteomic and transcriptomic analysis is useful to detect key molecular adaptation mechanisms of biotechnological interest for industrial processes. ILV5 gene seems to be important in wine yeast adaptation to low-temperature fermentation.   This study provides information that might help improve the future performance of wine yeast, either by genetic modification or by adaptation during industrial production. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  8. Rtm1: A Member of a New Family of Telomeric Repeated Genes in Yeast

    PubMed Central

    Ness, F.; Aigle, M.

    1995-01-01

    We have isolated a new yeast gene called RTM1 whose overexpression confers resistance to the toxicity of molasses. The RTM1 gene encodes a hydrophobic 34-kD protein that contains seven potential transmembrane-spanning segments. Analysis of a series of industrial strains shows that the sequence is present in multiple copies and in variable locations in the genome. RTM loci are always physically associated with SUC telomeric loci. The SUC-RTM sequences are located between X and Y' subtelomeric sequences at chromosome ends. Surprisingly RTM sequences are not detected in the laboratory strain X2180. The lack of this sequence is associated with the absence of any SUC telomeric gene previously described. This observation raises the question of the origin of this nonessential gene. The particular subtelomeric position might explain the SUC-RTM sequence amplification observed in the genome of yeasts used in industrial biomass or ethanol production with molasses as substrate. This SUC-RTM sequence dispersion seems to be a good example of genomic rearrangement playing a role in evolution and environmental adaptation in these industrial yeasts. PMID:7672593

  9. Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

    PubMed Central

    Conconi, Antonio; Bespalov, Vyacheslav A.; Smerdon, Michael J.

    2002-01-01

    Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. PMID:11782531

  10. Life-history strategies and carbon metabolism gene dosage in the Nakaseomyces yeasts.

    PubMed

    Legrand, Judith; Bolotin-Fukuhara, Monique; Bourgais, Aurélie; Fairhead, Cécile; Sicard, Delphine

    2016-03-01

    The Nakaseomyces clade consists of a group of six hemiascomyceteous yeasts (Candida glabrata, Nakaseomyces delphensis, C. nivarensis, C. bracarensis, C. castelli, N. bacillisporus), phylogenetically close to the yeast Saccharomyces cerevisiae, their representative being the well-known pathogenic yeast C. glabrata. Four species had been previously examined for their carbon assimilation properties and found to have similar properties to S. cerevisiae (repression of respiration in high glucose-i.e. Crabtree positivity-and being a facultative anaerobe). We examined here the complete set of the six species for their carbon metabolic gene content. We also measured different metabolic and life-history traits (glucose consumption rate, population growth rate, carrying capacity, cell size, cell and biomass yield). We observed deviations from the glycolytic gene redundancy observed in S. cerevisiae presumed to be an important property for the Crabtree positivity, especially for the two species C. castelli and N. bacillisporus which frequently have only one gene copy, but different life strategies. Therefore, we show that the decrease in carbon metabolic gene copy cannot be simply associated with a reduction of glucose consumption rate and can be counterbalanced by other beneficial genetic variations.

  11. Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast.

    PubMed

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

    2007-11-01

    Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.

  12. Diversity and antifungal susceptibility of yeasts from tropical freshwater environments in Southeastern Brazil.

    PubMed

    Medeiros, Adriana O; Kohler, Lidiane M; Hamdan, Junia S; Missagia, Beatriz S; Barbosa, Francisco A R; Rosa, Carlos A

    2008-08-01

    Yeast communities were isolated from water and sediment samples of two unpolluted natural lakes, located inside Rio Doce State Park, and two rivers located outside of this Park in Southeastern Brazil. A total of 134 yeast isolates were obtained and identified as belonging to 36 species. The numbers of fecal coliforms and yeast species were higher in rivers than in lakes. The genus Candida had the highest number of species with the presence of opportunistic pathogens such as Candida krusei, C. tropicalis, C. guilliermondii and C. parapsilosis. Yeasts able to grow at 37 degrees C were tested in relation to their susceptibility to common used antifungal drugs. Yeast isolates (13%) were susceptible to ketoconazole, 79% to fluconazole, 31% to terbinafine and 78% of the strains were susceptible to amphotericin B. Seven isolates from different Candida species were resistant to all antifungals tested. The high number of fecal coliforms found in these aquatic environments and the presence of resistant yeast strains to common used antifungal drugs suggest that these environments can pose potential health risks for people utilizing the contaminated waters.

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

    PubMed

    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.

  14. Yeast and bacterial diversity along a transect in an acidic, As-Fe rich environment revealed by cultural approaches.

    PubMed

    Delavat, François; Lett, Marie-Claire; Lièvremont, Didier

    2013-10-01

    Acid mine drainages (AMDs) are often thought to harbour low biodiversity, yet little is known about the diversity distribution along the drainages. Using culture-dependent approaches, the microbial diversity from the Carnoulès AMD sediment was investigated for the first time along a transect showing progressive environmental stringency decrease. In total, 20 bacterial genera were detected, highlighting a higher bacterial diversity than previously thought. Moreover, this approach led to the discovery of 16 yeast species, demonstrating for the first time the presence of this important phylogenetic group in this AMD. All in all, the location of the microbes along the transect helps to better understand their distribution in a pollution gradient. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Evolutionary History of Ascomyceteous Yeasts

    SciTech Connect

    Haridas, Sajeet; Riley, Robert; Salamov, Asaf; Goker, Markus; Klenk, Hans-Peter; Kurtzman, Cletus P.; Blackwell, Meredith; Grigoriev, Igor; Jeffries, Thomas W.

    2014-06-06

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with 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 with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.

  16. Yeast Reciprocal Hemizygosity to Confirm the Causality of a Quantitative Trait Loci-Associated Gene.

    PubMed

    Warringer, Jonas; Liti, Gianni; Blomberg, Anders

    2017-08-01

    Pinpointing causal alleles within a quantitative trait loci region is a key challenge when dissecting the genetic basis of natural variation. In yeast, homing in on culprit genes is often achieved using engineered reciprocal hemizygotes as outlined here. Based on prior information on gene-trait associations, candidate genes are identified. In haploid versions of both founder strains, a candidate gene is then deleted. Gene knockouts are independently mated to a wild-type version of the other strain, such that two diploid hybrid strains are obtained. These strains are identical with regard to the nuclear genome, except for that they are hemizygotic at the locus of interest and contain different alleles of the candidate gene. If correctly measured, a trait difference between these reciprocal hemizygotes can confidently be ascribed to allelic variation at the target locus. © 2017 Cold Spring Harbor Laboratory Press.

  17. Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

    PubMed Central

    Nakahara, Yoshiki; Sawabe, Shogo; Kainuma, Kenta; Katsuhara, Maki; Shibasaka, Mineo; Suzuki, Masanori; Yamamoto, Kosuke; Oguri, Suguru; Sakamoto, Hikaru

    2015-01-01

    Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation. PMID:26579166

  18. MATRIX FACTORIZATION-BASED DATA FUSION FOR GENE FUNCTION PREDICTION IN BAKER’S YEAST AND SLIME MOLD

    PubMed Central

    ŽITNIK, MARINKA; ZUPAN, BLAŽ

    2014-01-01

    The development of effective methods for the characterization of gene functions that are able to combine diverse data sources in a sound and easily-extendible way is an important goal in computational biology. We have previously developed a general matrix factorization-based data fusion approach for gene function prediction. In this manuscript, we show that this data fusion approach can be applied to gene function prediction and that it can fuse various heterogeneous data sources, such as gene expression profiles, known protein annotations, interaction and literature data. The fusion is achieved by simultaneous matrix tri-factorization that shares matrix factors between sources. We demonstrate the effectiveness of the approach by evaluating its performance on predicting ontological annotations in slime mold D. discoideum and on recognizing proteins of baker’s yeast S. cerevisiae that participate in the ribosome or are located in the cell membrane. Our approach achieves predictive performance comparable to that of the state-of-the-art kernel-based data fusion, but requires fewer data preprocessing steps. PMID:24297565

  19. Identification of genes responsible for improved cryoresistance in fermenting yeast cells.

    PubMed

    Tanghe, A; Teunissen, A; Van Dijck, P; Thevelein, J M

    2000-04-10

    Using repetitive freezing and thawing, different mutant industrial Saccharomyces cerevisiae strains with increased freeze resistance have been isolated. To get a better insight in the mechanisms responsible for this elevated resistance and to give us the opportunity to modify other strains so that they become more suitable for use in frozen dough preparations, we applied the microarray technology in order to identify genes that are differentially expressed in a freeze-resistant mutant when compared to a freeze-sensitive industrial yeast strain.

  20. A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

    DTIC Science & Technology

    2005-05-01

    cytoplasm and mitochondria METhionine requiring: Bisphosphate-3-nucleotidase, involved in salt tolerance and methionine biogenesis ; MET22 YOL064C... mitochondria ribosomal protein subunits (MRP and MRPL). These results suggest that slowing the growth rate of yeast by deleting genes in processes necessary...Putative sensor/transporter protein involved in cell wall biogenesis ; contains 14-16 transmembrane segments and 3several putative glycosylation and

  1. Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

    PubMed

    Peng, Huadong; Moghaddam, Lalehvash; Brinin, Anthony; Williams, Brett; Mundree, Sagadevan; Haritos, Victoria S

    2017-06-25

    As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  2. The yeast Dekkera bruxellensis genome contains two orthologs of the ARO10 gene encoding for phenylpyruvate decarboxylase.

    PubMed

    de Souza Liberal, Anna Theresa; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante; Simões, Diogo Ardaillon; de Morais, Marcos Antonio

    2012-07-01

    The yeast Dekkera bruxellensis possesses important physiological traits that enable it to grow in industrial environments as either spoiling yeast of wine production or a fermenting strain used for lambic beer, or fermenting yeast in the bioethanol production process. In this work, in silico analysis of the Dekkera genome database allowed the identification of two paralogous genes encoding for phenylpyruvate decarboxylase (DbARO10) that represents a unique trait among the hemiascomycetes. The molecular analysis of the theoretical protein confirmed its protein identity. Upon cultivation of the cell in medium containing phenylpyruvate, both increases in gene expression and in phenylpyruvate decarboxylase activity were observed. Both genes were differentially expressed depending on the culture condition and the type of metabolism, which indicated the difference in the biological function of their corresponding proteins. The importance of the duplicated DbARO10 genes in the D. bruxellensis genome was discussed and represents the first effort to understand the production of flavor by this yeast.

  3. Dissecting the regulation of yeast genes by the osmotin receptor

    PubMed Central

    Kupchak, Brian R.; Villa, Nancy Y.; Kulemina, Lidia; Lyons, Thomas J.

    2008-01-01

    The Izh2p protein from Saccharomyces cerevisiae is a receptor for the plant antifungal protein, osmotin. Since Izh2p is conserved in fungi, understanding its biochemical function could inspire novel strategies for the prevention of fungal growth. However, it has been difficult to determine the exact role of Izh2p because it has pleiotropic effects on cellular biochemistry. Herein, we demonstrate that Izh2p negatively regulates functionally divergent genes through a CCCTC promoter motif. Moreover, we show that Izh2p-dependent promoters containing this motif are regulated by the Nrg1p/Nrg2p and Msn2p/Msn4p transcription factors. The fact that Izh2p can regulate gene expression through this widely dispersed element presents a reasonable explanation of its pleiotropy. The involvement of Nrg1p/Nrgp2 in Izh2p-dependent gene regulation also suggests a role for this receptor in regulating fungal differentiation in response to stimuli produced by plants. PMID:18625204

  4. Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature

    PubMed Central

    López-Malo, María; García-Ríos, Estéfani; Chiva, Rosana; Guillamon, José M.

    2014-01-01

    Wine produced by low-temperature fermentation is mostly considered to have improved sensory qualities. However few commercial wine strains available on the market are well-adapted to ferment at low temperature (10 - 15°C). The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. One strategy to modify lipid composition is to alter transcriptional activity by deleting or overexpressing the key genes of lipid metabolism. In a previous study, we identified the genes of the phospholipid, sterol and sphingolipid pathways, which impacted on growth capacity at low temperature. In the present study, we aimed to determine the influence of these genes on fermentation performance and growth during low-temperature wine fermentations. We analyzed the phenotype during fermentation at the low and optimal temperature of the lipid mutant and overexpressing strains in the background of a derivative commercial wine strain. The increase in the gene dosage of some of these lipid genes, e.g., PSD1, LCB3, DPL1 and OLE1, improved fermentation activity during low-temperature fermentations, thus confirming their positive role during wine yeast adaptation to cold. Genes whose overexpression improved fermentation activity at 12°C were overexpressed by chromosomal integration into commercial wine yeast QA23. Fermentations in synthetic and natural grape must were carried out by this new set of overexpressing strains. The strains overexpressing OLE1 and DPL1 were able to finish fermentation before commercial wine yeast QA23. Only the OLE1 gene overexpression produced a specific aroma profile in the wines produced with natural grape must. PMID:28357215

  5. Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature.

    PubMed

    López-Malo, María; García-Ríos, Estéfani; Chiva, Rosana; Guillamon, José M

    2014-10-29

    Wine produced by low-temperature fermentation is mostly considered to have improved sensory qualities. However few commercial wine strains available on the market are well-adapted to ferment at low temperature (10 - 15°C). The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. One strategy to modify lipid composition is to alter transcriptional activity by deleting or overexpressing the key genes of lipid metabolism. In a previous study, we identified the genes of the phospholipid, sterol and sphingolipid pathways, which impacted on growth capacity at low temperature. In the present study, we aimed to determine the influence of these genes on fermentation performance and growth during low-temperature wine fermentations. We analyzed the phenotype during fermentation at the low and optimal temperature of the lipid mutant and overexpressing strains in the background of a derivative commercial wine strain. The increase in the gene dosage of some of these lipid genes, e.g., PSD1, LCB3, DPL1 and OLE1, improved fermentation activity during low-temperature fermentations, thus confirming their positive role during wine yeast adaptation to cold. Genes whose overexpression improved fermentation activity at 12°C were overexpressed by chromosomal integration into commercial wine yeast QA23. Fermentations in synthetic and natural grape must were carried out by this new set of overexpressing strains. The strains overexpressing OLE1 and DPL1 were able to finish fermentation before commercial wine yeast QA23. Only the OLE1 gene overexpression produced a specific aroma profile in the wines produced with natural grape must.

  6. Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans.

    PubMed

    Jespersen, Lene; Nielsen, Dennis S; Hønholt, Susanne; Jakobsen, Mogens

    2005-02-01

    Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.

  7. Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows.

    PubMed

    Jin, Dingxing; Kang, Kun; Wang, Hongze; Wang, Zhisheng; Xue, Bai; Wang, Lizhi; Xu, Feng; Peng, Quanhui

    2017-02-07

    This study aimed to investigate the effects of dietary supplementation of different dosages of active dried yeast (ADY) on the fecal methanogenic archaea community of dairy cattle. Twelve multiparous, healthy, mid-lactating Holstein dairy cows (body weight: 584 ± 23.2 kg, milk produced: 26.3 ± 1.22 kg/d) were randomly assigned to one of three treatments (control, ADY2, and ADY4) according to body weight with four replicates per treatment. Cows in the control group were fed conventional rations without ADY supplementation, while cows in the ADY2 and ADY4 group were fed rations supplemented with ADY at 2 or 4 g/d/head. Real-time PCR analysis showed the populations of total methanogens in the feces were significantly decreased (P < 0.05) in the ADY4 group compared with control. High-throughput sequencing technology was applied to examine the differences in methanogenic archaea diversity in the feces of the three treatment groups. A total of 155,609 sequences were recovered (a mean of 12,967 sequences per sample) from the twelve fecal samples, which consisted of a number of operational taxonomic units (OTUs) ranging from 1451 to 1,733, were assigned to two phyla, four classes, five orders, five families and six genera. Bioinformatic analyses illustrated that the natural fecal archaeal community of the control group was predominated by Methanobrevibacter (86.9% of the total sequence reads) and Methanocorpusculum (10.4%), while the relative abundance of the remaining four genera were below 1% with Methanosphaera comprising 0.8%, Thermoplasma composing 0.4%, and the relative abundance of Candidatus Nitrososphaera and Halalkalicoccus being close to zero. At the genus level, the relative abundances of Methanocorpusculum and Thermoplasma were increased (P < 0.05) with increasing dosage of ADY. Conversely, the predominant methanogen genus Methanobrevibacter was decreased with ADY dosage (P < 0.05). Dietary supplementation of ADY had no significant effect (P

  8. The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

    PubMed

    Hermann, G J; King, E J; Shaw, J M

    1997-04-07

    In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

  9. Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

    SciTech Connect

    Himelblau, E.; Amasino, R.M.; Mira, H.; Penarrubia, L.; Lin, S.J.; Culotta, V.C.

    1998-08-01

    A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and in fluorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO{sub 4}, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

  10. Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides

    PubMed Central

    Johns, Alexander M. B.; Love, John; Aves, Stephen J.

    2016-01-01

    Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides. PMID:27818654

  11. Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell.

    PubMed

    García, Ana M; Hernández, Orville; Aristizabal, Beatriz H; De Souza Bernardes, Luciano A; Puccia, Rosana; Naranjo, Tony W; Goldman, Gustavo H; Goldman, Maria H; Cano, Luz E; Restrepo, Angela; McEwen, Juan G

    2010-02-01

    Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence factors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40/79 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P. brasiliensis conidia induced infection.

  12. Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis1

    PubMed Central

    Himelblau, Edward; Mira, Helena; Lin, Su-Ju; Cizewski Culotta, Valeria; Peñarrubia, Lola; Amasino, Richard M.

    1998-01-01

    A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis. PMID:9701579

  13. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity

    PubMed Central

    Mohan, Vijee; Gupta, Soni; Thomas, Sherinmol; Mickey, Hanjabam; Charakana, Chaitanya; Chauhan, Vineeta Singh; Sharma, Kapil; Kumar, Rakesh; Tyagi, Kamal; Sarma, Supriya; Gupta, Suresh Kumar; Kilambi, Himabindu Vasuki; Nongmaithem, Sapana; Kumari, Alka; Gupta, Prateek; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima’D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future. PMID:27077652

  14. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

    PubMed

    Mohan, Vijee; Gupta, Soni; Thomas, Sherinmol; Mickey, Hanjabam; Charakana, Chaitanya; Chauhan, Vineeta Singh; Sharma, Kapil; Kumar, Rakesh; Tyagi, Kamal; Sarma, Supriya; Gupta, Suresh Kumar; Kilambi, Himabindu Vasuki; Nongmaithem, Sapana; Kumari, Alka; Gupta, Prateek; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

  15. Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

    PubMed Central

    Itoh, T; Ohtsuki, I; Yamashita, I; Fukui, S

    1987-01-01

    The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function. Images PMID:3114236

  16. Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast.

    PubMed Central

    Sone, H; Fujii, T; Kondo, K; Shimizu, F; Tanaka, J; Inoue, T

    1988-01-01

    The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production. PMID:3278689

  17. Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

    PubMed Central

    Vignais, M L; Woudt, L P; Wassenaar, G M; Mager, W H; Sentenac, A; Planta, R J

    1987-01-01

    Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:3301327

  18. Diversity of culturable yeasts associated with zoanthids from Brazilian reef and its relation with anthropogenic disturbance.

    PubMed

    Paulino, Gustavo Vasconcelos Bastos; Félix, Ciro Ramon; Broetto, Leonardo; Landell, Melissa Fontes

    2017-08-23

    Some of the main threats to coral reefs come from human actions on marine environment, such as tourism, overfishing and pollution from urban development. While several studies have demonstrated an association between bacteria and corals, demonstrating how these communities react to different anthropogenic stressors, yeast communities associated with corals have received far less attention from researchers. The aim of this work was therefore to describe cultivable yeasts associated with three coral species and to evaluate the influence of sewage discharge on yeasts community. We obtained 130 isolates, mostly belonging to phylum Ascomycota and many of them had previously been isolated from human samples or are considered pathogens. The mycobiota was more similar among corals collected from the same reef, indicating that the composition of reef yeast community is more influenced by environmental conditions than host species. We suggest further studies to elucidate which factors are most influential on the composition of the coral-associated yeast community. Copyright © 2017. Published by Elsevier Ltd.

  19. Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis.

    PubMed

    Piontek, M; Hagedorn, J; Hollenberg, C P; Gellissen, G; Strasser, A W

    1998-09-01

    Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression. The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comprise auxotrophic host strains (trp5 in the case of S. occidentalis; trp5-10, his3 in the case of P. stipitis) and suitable transformation vectors. Vector components consist of an S. occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host. A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species. Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S. occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.

  20. Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

    PubMed Central

    Clemens, S; Kim, E J; Neumann, D; Schroeder, J I

    1999-01-01

    Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes. PMID:10369673

  1. Transcript analysis of 250 novel yeast genes from chromosome XIV.

    PubMed

    Planta, R J; Brown, A J; Cadahia, J L; Cerdan, M E; de Jonge, M; Gent, M E; Hayes, A; Kolen, C P; Lombardia, L J; Sefton, M; Oliver, S G; Thevelein, J; Tournu, H; van Delft, Y J; Verbart, D J; Winderickx, J

    1999-03-15

    The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi-steady-state conditions (exponential growth on rich fermentative, rich non-fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo-stress, heat-shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACT1 mRNA. The levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift; five were repressed by glucose; six were induced in response to nitrogen starvation; three were induced in stationary phase; five were induced by osmo-stress; four were induced by heat-shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome-wide basis.

  2. One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha.

    PubMed

    González, C; Perdomo, G; Tejera, P; Brito, N; Siverio, J M

    1999-09-30

    Previous evidence based on the experience of our laboratory showed that one-step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one-step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR-mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed. Copyright 1999 John Wiley & Sons, Ltd.

  3. Analysis of yeast-like symbiote diversity in the brown planthopper (BPH), Nilaparvata lugens Stål, using a novel nested PCR-DGGE protocol.

    PubMed

    Hou, Yun; Ma, Zheng; Dong, Shengzhang; Chen, Yolanda H; Yu, Xiaoping

    2013-09-01

    Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S-ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S-ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC-ITS2, ITS1FGC-ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.

  4. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    PubMed

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  5. Molecular identification of yeasts associated with traditional Egyptian dairy products.

    PubMed

    El-Sharoud, W M; Belloch, C; Peris, D; Querol, A

    2009-09-01

    This study aimed to examine the diversity and ecology of yeasts associated with traditional Egyptian dairy products employing molecular techniques in yeast identification. A total of 120 samples of fresh and stored Domiati cheese, kariesh cheese, and "Matared" cream were collected from local markets and examined. Forty yeast isolates were cultured from these samples and identified using the restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region and sequencing of the domains D1 and D2 of the 26S rRNA gene. Yeasts were identified as Issatchenkia orientalis (13 isolates), Candida albicans (4 isolates), Clavispora lusitaniae (Candida lusitaniae) (9 isolates), Kodamaea ohmeri (Pichia ohmeri) (1 isolate), Kluyveromyces marxianus (6 isolates), and Candida catenulata (7 isolates). With the exception of C. lusitaniae, the D1/D2 26S rRNA gene sequences were 100% identical for the yeast isolates within the same species. Phylogenetic reconstruction of C. lusitaniae isolates grouped them into 3 distinguished clusters. Kariesh cheese was found to be the most diverse in its yeast floras and contained the highest total yeast count compared with other examined dairy products. This was linked to the acidic pH and lower salt content of this cheese, which favor the growth and survival of yeasts in foodstuffs. Stored Domiati cheese also contained diverse yeast species involving isolates of the pathogenic yeast C. albicans. This raises the possibility of dairy products being vehicles of transmission of pathogenic yeasts.

  6. Characterization of two genes required for the position-effect control of yeast mating-type genes.

    PubMed Central

    Shore, D; Squire, M; Nasmyth, K A

    1984-01-01

    The mating type of haploid yeast (a or alpha) is determined by information present at the MAT locus. Identical copies of a and alpha information are present at distal loci (HMR and HML), but transcription of these copies is repressed by the action, in trans, of four unlinked genes called SIR (silent information regulator). Repression by SIR also requires, in cis, DNA sequences called E which are found to the left of HML and HMR (but not MAT) and are greater than 1 kb from the mating-type gene promoters. SIR control can act on other promoters when they are brought near the E sequence, and thus the SIR gene products act in some general manner to repress transcription. We have determined the DNA sequence of two fragments which complement mutations in the SIR2 and SIR3 genes and show that these contain the structural genes by mapping the cloned sequences onto the yeast chromosome. The SIR2 and SIR3 coding sequences were identified by constructing gene disruptions and using these mutations to replace the normal chromosomal copies. Such null mutants of both SIR2 and SIR3 are defective in the position-effect control of the silent loci but have no other detectable phenotype. We have mapped the 5' and 3' ends of the SIR2 and SIR3 mRNAs and show that their level is unaffected by mutations in any of the four known SIR complementation groups. Images Fig. 2. Fig. 3. Fig. 4. PMID:6098447

  7. Gene duplication can impart fragility, not robustness, in the yeast protein interaction network.

    PubMed

    Diss, Guillaume; Gagnon-Arsenault, Isabelle; Dion-Coté, Anne-Marie; Vignaud, Hélène; Ascencio, Diana I; Berger, Caroline M; Landry, Christian R

    2017-02-10

    The maintenance of duplicated genes is thought to protect cells from genetic perturbations, but the molecular basis of this robustness is largely unknown. By measuring the interaction of yeast proteins with their partners in wild-type cells and in cells lacking a paralog, we found that 22 out of 56 paralog pairs compensate for the lost interactions. An equivalent number of pairs exhibit the opposite behavior and require each other's presence for maintaining their interactions. These dependent paralogs generally interact physically, regulate each other's abundance, and derive from ancestral self-interacting proteins. This reveals that gene duplication may actually increase mutational fragility instead of robustness in a large number of cases.

  8. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  9. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  10. Gene expression and biochemical analysis of cheese-ripening yeasts: focus on catabolism of L-methionine, lactate, and lactose.

    PubMed

    Cholet, Orianne; Hénaut, Alain; Casaregola, Serge; Bonnarme, Pascal

    2007-04-01

    DNA microarrays of 86 genes from the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Yarrowia lipolytica were developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of the LAC genes of K. marxianus was observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially in D. hansenii and K. marxianus, which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved in l-methionine catabolism was also used on the array. Y. lipolytica, which efficiently assimilates l-methionine, also exhibited a high expression of the Saccharomyces cerevisiae orthologs BAT2 and ARO8, which are involved in the l-methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures.

  11. An efficient method to isolate yeast genes causing overexpression-mediated growth arrest.

    PubMed

    Espinet, C; de la Torre, M A; Aldea, M; Herrero, E

    1995-01-01

    In order to characterize new yeast genes regulating cell proliferation, a number of overexpression-sensitive clones have been isolated from a Saccharomyces cerevisiae cDNA library in a multicopy vector under the control of the GAL1 promoter, on the basis of growth arrest phenotype under galactose-induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis-related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression-sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression of MCM1 (whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose-induced overexpression of MCM1 leads to rapid growth arrest at the G1 or S cell cycle stages, with many morphologically-abnormal cells. Several of the other clones also exhibit a G1 arrest terminal phenotype when overexpressed.

  12. An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes

    PubMed Central

    Kofoed, Megan; Milbury, Karissa L.; Chiang, Jennifer H.; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C.

    2015-01-01

    Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. PMID:26175450

  13. Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast.

    PubMed

    Trivedi, Dipesh Kumar; Yadav, Sandep; Vaid, Neha; Tuteja, Narendra

    2012-12-01

    Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins.

  14. Identification of bottom-fermenting yeast genes expressed during lager beer fermentation.

    PubMed

    Yoshida, Satoshi; Hashimoto, Kaori; Shimada, Emiko; Ishiguro, Tatsuji; Minato, Toshiko; Mizutani, Satoru; Yoshimoto, Hiroyuki; Tashiro, Kosuke; Kuhara, Satoru; Kobayashi, Osamu

    2007-07-01

    It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.

  15. Three distinct mechanisms of long-distance modulation of gene expression in yeast

    PubMed Central

    Du, Manyu; Zhang, Qian

    2017-01-01

    Recent Hi-C measurements have revealed numerous intra- and inter-chromosomal interactions in various eukaryotic cells. To what extent these interactions regulate gene expression is not clear. This question is particularly intriguing in budding yeast because it has extensive long-distance chromosomal interactions but few cases of gene regulation over-a-distance. Here, we developed a medium-throughput assay to screen for functional long-distance interactions that affect the average expression level of a reporter gene as well as its cell-to-cell variability (noise). We ectopically inserted an insulated MET3 promoter (MET3pr) flanked by ~1kb invariable sequences into thousands of genomic loci, allowing it to make contacts with different parts of the genome, and assayed the MET3pr activity in single cells. Changes of MET3pr activity in this case necessarily involve mechanisms that function over a distance. MET3pr has similar activities at most locations. However, at some locations, they deviate from the norm and exhibit three distinct patterns including low expression / high noise, low expression / low noise, and high expression / low noise. We provided evidence that these three patterns of MET3pr expression are caused by Sir2-mediated silencing, transcriptional interference, and 3D clustering. The clustering also occurs in the native genome and enhances the transcription of endogenous Met4-targeted genes. Overall, our results demonstrate that a small fraction of long-distance chromosomal interactions can affect gene expression in yeast. PMID:28426659

  16. Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast

    PubMed Central

    Trivedi, Dipesh Kumar; Yadav, Sandep; Vaid, Neha; Tuteja, Narendra

    2012-01-01

    Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins. PMID:23073011

  17. An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

    PubMed

    Kofoed, Megan; Milbury, Karissa L; Chiang, Jennifer H; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C

    2015-07-14

    Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.

  18. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    PubMed

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  19. ORF Organization and Gene Recognition in the Yeast Genome

    PubMed Central

    Li, Hong; Zhang, Lirong

    2003-01-01

    Some rules on gene recognition and ORF organization in the Saccharomyces cerevisiae genome are demonstrated by statistical analyses of sequence data. This study includes: (a) The random frame rule—that the six reading frames W1, W2, W3, C1, C2 and C3 in the double-stranded genome are randomly occupied by ORFs (related phenomena on ORF overlapping are also discussed). (b) The inhomogeneity rule—coding and non-coding ORFs differ in inhomogeneity of base composition in the three codon positions. By use of the inhomogeneity index (IHI), one can make a distinction between coding (IHI > 14) and non-coding (IHI ≤ 14) ORFs at 95% accuracy. We find that ‘spurious’ ORFs (with IHI ≤ 14) are distributed mainly in three classes of ORFs, namely, those with ‘similarity to unknown proteins’, those with ‘no similarity’, or ‘questionable ORFs’. The total number of spurious ORFs (which are unlikely to be regarded as coding ORFs) is estimated to be 470. (c) The evaluation of ORF length distribution shows that below 200 amino acids the occurrence of ATG initiator ORFs is close to random. PMID:18629282

  20. Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.

    PubMed

    Trikka, Fotini A; Nikolaidis, Alexandros; Athanasakoglou, Anastasia; Andreadelli, Aggeliki; Ignea, Codruta; Kotta, Konstantia; Argiriou, Anagnostis; Kampranis, Sotirios C; Makris, Antonios M

    2015-04-24

    Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation. Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.

  1. Effects of intense magnetic fields on sedimentation pattern and gene expression profile in budding yeast

    NASA Astrophysics Data System (ADS)

    Ikehata, Masateru; Iwasaka, Masakazu; Miyakoshi, Junji; Ueno, Shoogo; Koana, Takao

    2003-05-01

    Effects of magnetic fields (MFs) on biological systems are usually investigated using biological indices such as gene expression profiles. However, to precisely evaluate the biological effects of MF, the effects of intense MFs on systematic material transport processes including experimental environment must be seriously taken into consideration. In this study, a culture of the budding yeast, Saccharomyces cerevisiae, was used as a model for an in vitro biological test system. After exposure to 5 T static vertical MF, we found a difference in the sedimentation pattern of cells depending on the location of the dish in the magnet bore. Sedimented cells were localized in the center of the dish when they were placed in the lower part of the magnet bore while the sedimentation of the cells was uniform in dishes placed in the upper part of the bore because of the diamagnetic force. Genome wide gene expression profile of the yeast cells after exposure to 5 T static MF for 2 h suggested that the MF did not affect the expression level of any gene in yeast cells although the sedimentation pattern was altered. In addition, exposure to 10 T for 1 h and 5 T for 24 h also did not affect the gene expression. On the other hand, a slight change in expressions of several genes which are related to respiration was observed by exposure to a 14 T static MF for 24 h. The necessity of estimating the indirect effects of MFs on a study of its biological effect of MF in vitro will be discussed.

  2. Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method.

    PubMed

    Kappachery, Sajeesh; Yu, Jae Woong; Baniekal-Hiremath, Gangadhar; Park, Se Won

    2013-01-01

    Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato. © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  3. The enrichment of TATA box and the scarcity of depleted proximal nucleosome in the promoters of duplicated yeast genes.

    PubMed

    Kim, Yuseob; Lee, Jang H; Babbitt, Gregory A

    2010-01-01

    Population genetic theory of gene duplication suggests that the preservation of duplicate copies requires functional divergence upon duplication. Genes that can be readily modified to produce new gene expression patterns may thus be duplicated often. In yeast, genes exhibit dichotomous expression patterns based on their promoter architectures. The expression of genes that contain TATA box or occupied proximal nucleosome (OPN) tends to be variable and respond to external signals. On the other hand, genes without TATA box or with depleted proximal nucleosome (DPN) are expressed constitutively. We find that recent duplicates in the yeast genome are heavily biased to be TATA box containing genes and not to be DPN genes. This suggests that variably expressed genes, due to the functional organization in their promoters, have higher duplicability than constitutively expressed genes.

  4. Evaluation of microbial quality and yeast diversity in fresh-cut apple.

    PubMed

    Graça, Ana; Santo, David; Esteves, Eduardo; Nunes, Carla; Abadias, Maribel; Quintas, Célia

    2015-10-01

    The present work's aim was to study the microbial quality of minimally processed apples commercialized in Portugal. Sixty eight samples of fresh-cut apple were analyzed before their best-before date in 2011 and 2012 for aerobic mesophilic and psychrotrophic microorganisms, total coliforms, lactic-acid bacteria (LAB), coagulase-positive staphylococci and fungi. The parameters of food safety studied were Cronobacter sakazakii, Salmonella spp. and Listeria sp. Samples were analyzed according to standard methodologies and using Chromocult Agar for coliforms and Escherichia coli. The yeasts were identified by restriction analysis of the ITS-5.8S rDNA-region and 26S rDNA partial sequencing. The mesophilic and psychrotrophic microorganisms ranged from 3.3 to 8.9 and from 4.9 to 8.4 log CFU/g, respectively. Coliforms were detected in all the samples and staphylococci in 5.8% of them. LAB numbers varied from 2.8 to 8.7 and fungi (yeast and molds) from 3.6 to 7.1 log CFU/g. The most common yeasts were Candida sake and Pichia fermentans followed by Hanseniaspora spp., Candida spp., Meyerozyma guilliermondii, Metschnikowia pulcherrima, Cryptococcus spp. and the psychrotrophic Cystofilobasidium infirmominiatum. Foodborne bacteria and opportunistic pathogenic yeasts were not detected in the apples studied. The results obtained respected the European Commission regulation regarding criteria of food hygiene and safety.

  5. Diversity and molecular determination of wild yeasts in a central Washington vineyard

    USDA-ARS?s Scientific Manuscript database

    Yeasts were isolated from grapes collected from a research vineyard at the WSU IAREC, located at Prosser, WA. Species determination was based on cultural features, microscopic morphology, physiological tests and analysis of ITS and D1/D2 rDNA sequence data. 53 species were found distributed among fi...

  6. The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination

    PubMed Central

    Viel, Alessia; Legras, Jean-Luc; Nadai, Chiara; Carlot, Milena; Lombardi, Angiolella; Crespan, Manna; Migliaro, Daniele; Giacomini, Alessio; Corich, Viviana

    2017-01-01

    In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota. PMID:28883812

  7. The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination.

    PubMed

    Viel, Alessia; Legras, Jean-Luc; Nadai, Chiara; Carlot, Milena; Lombardi, Angiolella; Crespan, Manna; Migliaro, Daniele; Giacomini, Alessio; Corich, Viviana

    2017-01-01

    In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota.

  8. Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.

    PubMed

    Wang, H; Jones, R W

    1999-09-01

    Functional expression of a beta-D-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60 degrees C, whereas the enzyme activity was optimal at 40 degrees C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.

  9. Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri.

    PubMed

    Turakainen, Hilkka; Korhola, Matti

    2005-07-30

    We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative beta-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.

  10. Cell-cycle control of gene expression in budding and fission yeast.

    PubMed

    Bähler, Jürg

    2005-01-01

    Cell-cycle control of transcription seems to be a universal feature of proliferating cells, although relatively little is known about its biological significance and conservation between organisms. The two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have provided valuable complementary insight into the regulation of periodic transcription as a function of the cell cycle. More recently, genome-wide studies of proliferating cells have identified hundreds of periodically expressed genes and underlying mechanisms of transcriptional control. This review discusses the regulation of three major transcriptional waves, which roughly coincide with three main cell-cycle transitions (initiation of DNA replication, entry into mitosis, and exit from mitosis). I also compare and contrast the transcriptional regulatory networks between the two yeasts and discuss the evolutionary conservation and possible roles for cell cycle-regulated transcription.

  11. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

    PubMed Central

    Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

  12. Systematic identification of gene annotation errors in the widely used yeast mutation collections.

    PubMed

    Ben-Shitrit, Taly; Yosef, Nir; Shemesh, Keren; Sharan, Roded; Ruppin, Eytan; Kupiec, Martin

    2012-02-05

    The baker's yeast mutation collections are extensively used genetic resources that are the basis for many genome-wide screens and new technologies. Anecdotal evidence has previously pointed to the putative existence of a neighboring gene effect (NGE) in these collections. NGE occurs when the phenotype of a strain carrying a particular perturbed gene is due to the lack of proper function of its adjacent gene. Here we performed a large-scale study of NGEs, presenting a network-based algorithm for detecting NGEs and validating software predictions using complementation experiments. We applied our approach to four datasets uncovering a similar magnitude of NGE in each (7-15%). These results have important consequences for systems biology, as the mutation collections are extensively used in almost every aspect of the field, from genetic network analysis to functional gene annotation.

  13. Cell cycle Start is coupled to entry into the yeast metabolic cycle across diverse strains and growth rates

    PubMed Central

    Burnetti, Anthony J.; Aydin, Mert; Buchler, Nicolas E.

    2016-01-01

    Cells have evolved oscillators with different frequencies to coordinate periodic processes. Here we studied the interaction of two oscillators, the cell division cycle (CDC) and the yeast metabolic cycle (YMC), in budding yeast. Previous work suggested that the CDC and YMC interact to separate high oxygen consumption (HOC) from DNA replication to prevent genetic damage. To test this hypothesis, we grew diverse strains in chemostat and measured DNA replication and oxygen consumption with high temporal resolution at different growth rates. Our data showed that HOC is not strictly separated from DNA replication; rather, cell cycle Start is coupled with the initiation of HOC and catabolism of storage carbohydrates. The logic of this YMC–CDC coupling may be to ensure that DNA replication and cell division occur only when sufficient cellular energy reserves have accumulated. Our results also uncovered a quantitative relationship between CDC period and YMC period across different strains. More generally, our approach shows how studies in genetically diverse strains efficiently identify robust phenotypes and steer the experimentalist away from strain-specific idiosyncrasies. PMID:26538026

  14. Cell cycle Start is coupled to entry into the yeast metabolic cycle across diverse strains and growth rates.

    PubMed

    Burnetti, Anthony J; Aydin, Mert; Buchler, Nicolas E

    2016-01-01

    Cells have evolved oscillators with different frequencies to coordinate periodic processes. Here we studied the interaction of two oscillators, the cell division cycle (CDC) and the yeast metabolic cycle (YMC), in budding yeast. Previous work suggested that the CDC and YMC interact to separate high oxygen consumption (HOC) from DNA replication to prevent genetic damage. To test this hypothesis, we grew diverse strains in chemostat and measured DNA replication and oxygen consumption with high temporal resolution at different growth rates. Our data showed that HOC is not strictly separated from DNA replication; rather, cell cycle Start is coupled with the initiation of HOC and catabolism of storage carbohydrates. The logic of this YMC-CDC coupling may be to ensure that DNA replication and cell division occur only when sufficient cellular energy reserves have accumulated. Our results also uncovered a quantitative relationship between CDC period and YMC period across different strains. More generally, our approach shows how studies in genetically diverse strains efficiently identify robust phenotypes and steer the experimentalist away from strain-specific idiosyncrasies. © 2016 Burnetti et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Life History Responses and Gene Expression Profiles of the Nematode Pristionchus pacificus Cultured on Cryptococcus Yeasts

    PubMed Central

    Sanghvi, Gaurav V.; Baskaran, Praveen; Röseler, Waltraud; Sieriebriennikov, Bogdan; Rödelsperger, Christian; Sommer, Ralf J.

    2016-01-01

    Nematodes, the earth’s most abundant metazoa are found in all ecosystems. In order to survive in diverse environments, they have evolved distinct feeding strategies and they can use different food sources. While some nematodes are specialists, including parasites of plants and animals, others such as Pristionchus pacificus are omnivorous feeders, which can live on a diet of bacteria, protozoans, fungi or yeast. In the wild, P. pacificus is often found in a necromenic association with beetles and is known to be able to feed on a variety of microbes as well as on nematode prey. However, in laboratory studies Escherichia coli OP50 has been used as standard food source, similar to investigations in Caenorhabditis elegans and it is unclear to what extent this biases the obtained results and how relevant findings are in real nature. To gain first insight into the variation in traits induced by a non-bacterial food source, we study Pristionchus-fungi interactions under laboratory conditions. After screening different yeast strains, we were able to maintain P. pacificus for at least 50–60 generations on Cryptococcus albidus and Cryptococcus curvatus. We describe life history traits of P. pacificus on both yeast strains, including developmental timing, survival and brood size. Despite a slight developmental delay and problems to digest yeast cells, which are both reflected at a transcriptomic level, all analyses support the potential of Cryptococcus strains as food source for P. pacificus. In summary, our work establishes two Cryptococcus strains as alternative food source for P. pacificus and shows change in various developmental, physiological and morphological traits, including the transcriptomic profiles. PMID:27741297

  16. Life History Responses and Gene Expression Profiles of the Nematode Pristionchus pacificus Cultured on Cryptococcus Yeasts.

    PubMed

    Sanghvi, Gaurav V; Baskaran, Praveen; Röseler, Waltraud; Sieriebriennikov, Bogdan; Rödelsperger, Christian; Sommer, Ralf J

    2016-01-01

    Nematodes, the earth's most abundant metazoa are found in all ecosystems. In order to survive in diverse environments, they have evolved distinct feeding strategies and they can use different food sources. While some nematodes are specialists, including parasites of plants and animals, others such as Pristionchus pacificus are omnivorous feeders, which can live on a diet of bacteria, protozoans, fungi or yeast. In the wild, P. pacificus is often found in a necromenic association with beetles and is known to be able to feed on a variety of microbes as well as on nematode prey. However, in laboratory studies Escherichia coli OP50 has been used as standard food source, similar to investigations in Caenorhabditis elegans and it is unclear to what extent this biases the obtained results and how relevant findings are in real nature. To gain first insight into the variation in traits induced by a non-bacterial food source, we study Pristionchus-fungi interactions under laboratory conditions. After screening different yeast strains, we were able to maintain P. pacificus for at least 50-60 generations on Cryptococcus albidus and Cryptococcus curvatus. We describe life history traits of P. pacificus on both yeast strains, including developmental timing, survival and brood size. Despite a slight developmental delay and problems to digest yeast cells, which are both reflected at a transcriptomic level, all analyses support the potential of Cryptococcus strains as food source for P. pacificus. In summary, our work establishes two Cryptococcus strains as alternative food source for P. pacificus and shows change in various developmental, physiological and morphological traits, including the transcriptomic profiles.

  17. Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure.

    PubMed Central

    Wilson, D W; Meacock, P A

    1988-01-01

    Strains of the yeast Kluyveromyces lactis that produce killer-toxin have been found to contain two linear dsDNA plasmids, k1 (8.9 Kb) and k2 (13.4 Kb). The four transcribed open reading frames of plasmid k1 contain no recognisable yeast nuclear expression signals. Moreover, a toxin subunit gene fused with the lacZ gene of Escherichia coli is not detectably expressed when introduced to K.lactis or Saccharomyces cerevisiae on a nuclear vector, even when native k1 and k2 are present in the cell. This and other evidence is consistent with the hypothesis that k1 and k2 reside in an extranuclear location, and do not utilise the nuclear RNA polymerases I, II or III for transcription of their genes. Sequencing of plasmid k2, which is thought to encode factors necessary for the maintenance or expression of k1, reveals an open reading frame predicted to encode a 974 amino acid polypeptide with homology to several DNA-directed RNA polymerases. We suggest that this is a component of a novel plasmid-specific extranuclear gene expression system. PMID:3138657

  18. Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

    PubMed Central

    Kitamoto, K; Oda, K; Gomi, K; Takahashi, K

    1991-01-01

    Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase. Images PMID:2036017

  19. A dynamin-like protein encoded by the yeast sporulation gene SPO15.

    PubMed

    Yeh, E; Driscoll, R; Coltrera, M; Olins, A; Bloom, K

    1991-02-21

    The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae. Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles. Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body. Thus cells are unable to form a bipolar spindle. Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids. This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin. The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules. SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.

  20. Global changes in gene expression of grapefruit peel tissue in response to the yeast biocontrol agent Metschnikowia fructicola.

    PubMed

    Hershkovitz, Vera; Ben-Dayan, Clarita; Raphael, Ginat; Pasmanik-Chor, Metsada; Liu, Jia; Belausov, Eduard; Aly, Radi; Wisniewski, Michael; Droby, Samir

    2012-05-01

    To gain a better understanding of the molecular changes taking place in citrus fruit tissue following the application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. Using a cut-off of P < 0.05 and a 1.5-fold change difference as biologically significant, the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. Microarray results of selected genes were validated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The data indicated that yeast application induced the expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. Moreover, suppression was correlated with significantly higher levels of hydrogen peroxide, superoxide anion and hydroxyl radical production in yeast-treated surface wounds. Interestingly, large amounts of hydrogen peroxide were detected inside yeast cells recovered from wounded fruit tissue, indicating the ability of the yeast to activate reactive oxygen species when it is in contact with plant tissue. This study provides the first global picture of gene expression changes in grapefruit in response to the yeast antagonist M. fructicola. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  1. Phylogenomic analyses reveal the diversity of laccase-coding genes in Fonsecaea genomes.

    PubMed

    Moreno, Leandro Ferreira; Feng, Peiying; Weiss, Vinicius Almir; Vicente, Vania Aparecida; Stielow, J Benjamin; de Hoog, Sybren

    2017-01-01

    The genus Fonsecaea comprises black yeast-like fungi of clinical relevance, including etiologic agents of chromoblastomycosis and cerebral phaeohyphomycosis. Presence of melanin and assimilation of monoaromatic hydrocarbons and alkylbenzenes have been proposed as virulence factors. Multicopper oxidase (MCO) is a family of enzymes including laccases, ferroxidases and ascorbate oxidases which are able to catalyze the oxidation of various aromatic organic compounds with the reduction of molecular oxygen to water. Additionally, laccases are required for the production of fungal melanins, a cell-wall black pigment recognized as a key polymer for pathogenicity and extremotolerance in black yeast-like fungi. Although the activity of laccase enzymes has previously been reported in many wood-rotting fungi, the diversity of laccase genes in Fonsecaea has not yet been assessed. In this study, we identified and characterized laccase-coding genes and determined their genomic location in five clinical and environmental Fonsecaea species. The identification of laccases sensu stricto will provide insights into carbon acquisition strategies as well as melanin production in Fonsecaea.

  2. Phylogenomic analyses reveal the diversity of laccase-coding genes in Fonsecaea genomes

    PubMed Central

    Feng, Peiying; Weiss, Vinicius Almir; Vicente, Vania Aparecida; Stielow, J. Benjamin; de Hoog, Sybren

    2017-01-01

    The genus Fonsecaea comprises black yeast-like fungi of clinical relevance, including etiologic agents of chromoblastomycosis and cerebral phaeohyphomycosis. Presence of melanin and assimilation of monoaromatic hydrocarbons and alkylbenzenes have been proposed as virulence factors. Multicopper oxidase (MCO) is a family of enzymes including laccases, ferroxidases and ascorbate oxidases which are able to catalyze the oxidation of various aromatic organic compounds with the reduction of molecular oxygen to water. Additionally, laccases are required for the production of fungal melanins, a cell-wall black pigment recognized as a key polymer for pathogenicity and extremotolerance in black yeast-like fungi. Although the activity of laccase enzymes has previously been reported in many wood-rotting fungi, the diversity of laccase genes in Fonsecaea has not yet been assessed. In this study, we identified and characterized laccase-coding genes and determined their genomic location in five clinical and environmental Fonsecaea species. The identification of laccases sensu stricto will provide insights into carbon acquisition strategies as well as melanin production in Fonsecaea. PMID:28187150

  3. Diversity and Physiological Characterization of D-Xylose-Fermenting Yeasts Isolated from the Brazilian Amazonian Forest

    PubMed Central

    Cadete, Raquel M.; Melo, Monaliza A.; Dussán, Kelly J.; Rodrigues, Rita C. L. B.; Silva, Silvio S.; Zilli, Jerri E.; Vital, Marcos J. S.; Gomes, Fátima C. O.; Lachance, Marc-André; Rosa, Carlos A.

    2012-01-01

    Background This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L·h to 0.75 g/L·h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L·h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates. PMID:22912807

  4. The mouse Enhancer trap locus 1 (Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes.

    PubMed

    Soininen, R; Schoor, M; Henseling, U; Tepe, C; Kisters-Woike, B; Rossant, J; Gossler, A

    1992-11-01

    A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a lacZ enhancer trap integration in the mouse genome showing a specific beta-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2/SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation and/or influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development.

  5. APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast.

    PubMed

    Saini, Natalie; Roberts, Steven A; Sterling, Joan F; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2017-05-01

    Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis. Published by Elsevier B.V.

  6. Disparate evolution of yeasts and filamentous fungi indicated by phylogenetic analysis of glyceraldehyde-3-phosphate dehydrogenase genes.

    PubMed Central

    Smith, T L

    1989-01-01

    Genes encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from several evolutionarily disparate organisms were used to construct a phylogenetic tree by evolutionary parsimony. The GAPDH tree indicates that, in contrast to the presently accepted taxonomy of fungi, the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii evolved separately from the filamentous ascomycetes (such as Aspergillus nidulans) with which these yeasts are classified. According to this tree, the Saccharomyces-like yeasts evolved very early in the course of eukaryotic evolution, whereas both ascomycete and basidiomycete filamentous fungi diverged much later through a common ancestor. PMID:2674943

  7. Winemaking and Bioprocesses Strongly Shaped the Genetic Diversity of the Ubiquitous Yeast Torulaspora delbrueckii

    PubMed Central

    Comte, Guillaume; Panfili, Aurélie; Delcamp, Adline; Salin, Franck; Marullo, Philippe; Bely, Marina

    2014-01-01

    The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼1900 years ago), and to the Neolithic era for bioprocesses (∼4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses. PMID:24718638

  8. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks

    PubMed Central

    Zhou, Chunshui; Elia, Andrew E. H.; Naylor, Maria L.; Ballif, Bryan A.; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M.; Xavier, Ramnik J.; Gygi, Steven P.; Elledge, Stephen J.

    2016-01-01

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  9. Diversity and activities of yeasts from different parts of a Stilton cheese.

    PubMed

    Gkatzionis, Konstantinos; Yunita, Dewi; Linforth, Robert S T; Dickinson, Matthew; Dodd, Christine E R

    2014-05-02

    Blue cheeses are very complex food matrices presenting significant spatial differentiation between sections and the Stilton variety also has a hard brown crust making its matrix even more complex. The mycobiota communities in the three sections (blue veins, white core and outer crust) of a Stilton blue cheese were studied by employing culture-independent (TRFLP, DGGE) and culture-dependent analyses. Yeasts isolated from the cheese were studied for aroma production in a dairy model system with and without the starter Lactococcus lactis and filamentous fungus Penicillium roqueforti using SPME GC-MS. Significant qualitative and quantitative differences were observed in the yeast communities between the cheese sections with all the techniques. Yarrowia lipolytica presented strong synergistic activity with P. roqueforti enhancing the production of ketone aroma compounds, characteristic of blue cheeses. Culture techniques allowed the observation of the presence and uneven distribution of two different morphological groups of Debaryomyces hansenii in the different sections and of Trichosporon ovoides but failed to isolate Candida catenulata which dominated some parts of the cheese in the culture-independent analysis. This suggests that this species may be an important early coloniser but fails to survive into the final cheese. The study indicated that the yeast flora in the cheese sections differ including isolates that could affect their aroma profiles.

  10. Diversity of yeast and mold species from a variety of cheese types.

    PubMed

    Banjara, Nabaraj; Suhr, Mallory J; Hallen-Adams, Heather E

    2015-06-01

    To generate a comprehensive profile of viable fungi (yeasts and molds) on cheese as it is purchased by consumers, 44 types of cheese were obtained from a local grocery store from 1 to 4 times each (depending on availability) and sampled. Pure cultures were obtained and identified by DNA sequence of the ITS region, as well as growth characteristics and colony morphology. The yeast Debaryomyces hansenii was the most abundant fungus, present in 79 % of all cheeses and 63 % of all samples. Penicillium roqueforti was the most common mold, isolated from a variety of cheeses in addition to the blue cheeses. Eighteen other fungal species were isolated, ten from only one sample each. Most fungi isolated have been documented from dairy products; a few raise potential food safety concerns (i.e. Aspergillus flavus, isolated from a single sample and capable of producing aflatoxins; and Candida parapsilosis, an emerging human pathogen isolated from three cheeses). With the exception of D. hansenii (present in most cheese) and P. roqueforti (a necessary component of blue cheese), no strong correlation was observed between cheese type, manufacturer, or sampling time with the yeast or mold species composition.

  11. Evaluation of fungal and yeast diversity in Slovakian wine-related microbial communities.

    PubMed

    Brežná, Barbara; Zenišová, Katarína; Chovanová, Katarína; Chebeňová, Viera; Kraková, Lucia; Kuchta, Tomáš; Pangallo, Domenico

    2010-11-01

    Since the yeast flora of Slovakian enology has not previously been investigated by culture-independent methods, this approach was applied to two most common cultivars Frankovka (red wine) and Veltlin (white wine), and complemented by cultivation. Model samples included grapes, initial must, middle fermenting must and must in the end-fermentation phase. The cultured isolates were characterized by length polymorphism of rDNA spacer two region using fluorescence PCR and capillary electrophoresis (f-ITS PCR), and some were identified by sequencing. The microbial DNA extracted directly from the samples without cultivation was analysed by f-ITS PCR, amplicons were cloned and sequenced. The use of universal fungal primers led to detection of both yeasts and filamentous fungi. The amplicon of highest intensity and present in all the samples corresponded to Hanseniaspora uvarum. Other species demonstrated by both approaches included Saccharomyces sp., Metschnikowia pulcherrima or M. chrysoperlae, Candida zemplinina, Cladosporium cladosporioides, Botryotinia fuckeliana, Pichia anomala, Candida railenensis, Cryptococcus magnus, Metschnikowia viticola or Candida kofuensis, Pichia kluyveri or Pichia fermentas, Pichia membranifaciens, Aureobasidium pullulans, Alternaria alternata, Erysiphe necator, Rhodotorula glutinis, Issatchenkia terricola and Debaryomyces hansenii. Endemism of Slovakian enological yeasts was suggested on the level of minor genetic variations of the known species and probably not accounting for novel species. The prevalence of H. uvarum over Saccharomyces sp. in the samples was indicated. This is the first culture-independent study of Slovakian enology and the first time f-ITS PCR profiling was used on wine-related microbial communities.

  12. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    PubMed

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

  13. The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

    PubMed Central

    Hermann, Greg J.; King, Edward J.; Shaw, Janet M.

    1997-01-01

    In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043

  14. [Effects of knockout ECM25/YJL201W gene in brewing yeast on beer flavor stability].

    PubMed

    Zhang, Yixin; Li, Qi; Shen, Wei; Xie, Yan; Gu, Guoxian

    2008-08-01

    The ECM25 deletion mutant of industrial brewing yeast, G03/a, was constructed by replacing the ECM25 gene with the kanMX gene. The transformant was verified to be genetically stable. The PCR analysis showed that ECM25 gene in the G-03/a was deleted. Under aerobic conditions of ll degrees C and 28 degrees C, compared with the host strain G-03, the excretive glutathione concentration of G-03/a increased by 21.4% and 14.7%, respectively. Strains G-03 and G-03/a were inoculated in flasks and cultivated continuously for 4 generations. Compared with the host strain G-03, the glutathione concentration in the main fermentation broth and final beer of strain G-03/a increased by 32.1% and 13.8%, the stability index (SI) increased by 7.7% and 5.3%, respectively, and the flavor resistance staling value (RSV value) in final beer increased by 45.0%. During EBC fermentation, the glutathione concentration in the main fermentation broth of strain G-03/a increased by 34.0%, compared with the host strain G-03. Furthermore, no significant difference in routine fermentation parameters was found. The strain G-03/a is proved to be an excellent anti-staling brewing yeast to improve beer flavor stability.

  15. Yeast adapts to a changing stressful environment by evolving cross-protection and anticipatory gene regulation.

    PubMed

    Dhar, Riddhiman; Sägesser, Rudolf; Weikert, Christian; Wagner, Andreas

    2013-03-01

    Organisms can protect themselves against future environmental change. An example is cross-protection, where physiological adaptation against a present environmental stressor can protect an organism against a future stressor. Another is anticipation, where an organism uses information about its present environment to trigger gene expression and other physiological changes adaptive in future environments. "Predictive" abilities like this exist in organisms that have been exposed to periodic changes in environments. It is unknown how readily they can evolve. To answer this question, we carried out laboratory evolution experiments in the yeast Saccharomyces cerevisiae. Specifically, we exposed three replicate populations of yeast to environments that varied cyclically between two stressors, salt stress and oxidative stress, every 10 generations, for a total of 300 generations. We evolved six replicate control populations in only one of these stressors for the same amount of time. We analyzed fitness changes and genome-scale expression changes in all these evolved populations. Our populations evolved asymmetric cross protection, where oxidative stress protects against salt stress but not vice versa. Gene expression data also suggest the evolution of anticipation and basal gene expression changes that occur uniquely in cyclic environments. Our study shows that highly complex physiological states that are adaptive in future environments can evolve on very short evolutionary time scales.

  16. Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

    PubMed

    Rao, Suryadevara S; Hildebrand, David

    2009-10-01

    The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

  17. Transfer RNA Genes Are Genomic Targets for De Novo Transposition of the Yeast Retrotransposon Ty3

    PubMed Central

    Chalker, D. L.; Sandmeyer, S. B.

    1990-01-01

    Insertions of the yeast element Ty3 resulting from induced retrotransposition were characterized in order to identify the genomic targets of transposition. The DNA sequences of the junctions between Ty3 and flanking DNA were determined for two insertions of an unmarked element. Each insertion was at position -17 from the 5' end of a tRNA-coding sequence. Ninety-one independent insertions of a marked Ty3 element were studied by Southern blot analysis. Pairs of independent insertions into seven genomic loci accounted for 14 of these insertions. The DNA sequence flanking the insertion site was determined for at least one member of each pair of integrated elements. In each case, insertion was at position -16 or -17 relative to the 5' end of one of seven different tRNA genes. This proportion of genomic loci used twice for Ty3 integration is consistent with that predicted by a Poisson distribution for a number of genomic targets roughly equivalent to the estimated number of yeast tRNA genes. In addition, insertions upstream of the same tRNA gene in one case were at different positions, but in all cases were in the same orientation. Thus, genomic insertions of Ty3 in a particular orientation are apparently specified by the target, while the actual position of the insertion relative to the tRNA-coding sequence can vary slightly. PMID:1963869

  18. In silico design and in vivo implementation of yeast gene Boolean gates

    PubMed Central

    2014-01-01

    In our previous computational work, we showed that gene digital circuits can be automatically designed in an electronic fashion. This demands, first, a conversion of the truth table into Boolean formulas with the Karnaugh map method and, then, the translation of the Boolean formulas into circuit schemes organized into layers of Boolean gates and Pools of signal carriers. In our framework, gene digital circuits that take up to three different input signals (chemicals) arise from the composition of three kinds of basic Boolean gates, namely YES, NOT, and AND. Here we present a library of YES, NOT, and AND gates realized via plasmidic DNA integration into the yeast genome. Boolean behavior is reproduced via the transcriptional control of a synthetic bipartite promoter that contains sequences of the yeast VPH1 and minimal CYC1 promoters together with operator binding sites for bacterial (i.e. orthogonal) repressor proteins. Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required. Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits. As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library. PMID:24485181

  19. Yeast mutant affected for viability upon nutrient starvation: characterization and cloning of the RVS161 gene.

    PubMed

    Crouzet, M; Urdaci, M; Dulau, L; Aigle, M

    1991-10-01

    In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditions have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161-1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.

  20. In silico design and in vivo implementation of yeast gene Boolean gates.

    PubMed

    Marchisio, Mario A

    2014-02-02

    In our previous computational work, we showed that gene digital circuits can be automatically designed in an electronic fashion. This demands, first, a conversion of the truth table into Boolean formulas with the Karnaugh map method and, then, the translation of the Boolean formulas into circuit schemes organized into layers of Boolean gates and Pools of signal carriers. In our framework, gene digital circuits that take up to three different input signals (chemicals) arise from the composition of three kinds of basic Boolean gates, namely YES, NOT, and AND. Here we present a library of YES, NOT, and AND gates realized via plasmidic DNA integration into the yeast genome. Boolean behavior is reproduced via the transcriptional control of a synthetic bipartite promoter that contains sequences of the yeast VPH1 and minimal CYC1 promoters together with operator binding sites for bacterial (i.e. orthogonal) repressor proteins. Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required. Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits. As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library.

  1. Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA.

    PubMed Central

    Foury, F; Lahaye, A

    1987-01-01

    The nuclear gene PIF of Saccharomyces cerevisiae is required for both repair of mitochondrial DNA (mtDNA) and recognition of a recombinogenic signal characterized by a 26-bp palindromic AT sequence in the ery region of mtDNA. This gene has been cloned in yeast by genetic complementation of pif mutants. Its chromosomal disruption does not destroy the genetic function of mitochondria. The nucleotide sequence of the 3.5-kb insert from a complementing plasmid reveals an open reading frame encoding a potential protein of 857 amino acids and Mr = 97,500. An ATP-binding domain is present in the central part of the gene and in the carboxy-terminal region a putative DNA-binding site is present. Its alpha helix-turn-alpha helix motif is found in DNA-binding proteins such as lambda and lactose repressors which recognize symmetric sequences. Significant amino acid homology is observed with yeast RAD3 and E. coli UvrD (helicase II) proteins which are required for excision repair of damaged DNA. Images Fig. 1. Fig. 2. PMID:3038524

  2. The Awa1 gene is required for the foam-forming phenotype and cell surface hydrophobicity of sake yeast.

    PubMed

    Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

    2002-04-01

    Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.

  3. The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

    PubMed Central

    Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

    2002-01-01

    Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast. PMID:11916725

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

    PubMed

    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.

  5. The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components.

    PubMed

    Stirling, Peter C; Bloom, Michelle S; Solanki-Patil, Tejomayee; Smith, Stephanie; Sipahimalani, Payal; Li, Zhijian; Kofoed, Megan; Ben-Aroya, Shay; Myung, Kyungjae; Hieter, Philip

    2011-04-01

    Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼ 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.

  6. The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

    PubMed Central

    Stirling, Peter C.; Bloom, Michelle S.; Solanki-Patil, Tejomayee; Smith, Stephanie; Sipahimalani, Payal; Li, Zhijian; Kofoed, Megan; Ben-Aroya, Shay; Myung, Kyungjae; Hieter, Philip

    2011-01-01

    Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease. PMID:21552543

  7. [Soil microbial ecological process and microbial functional gene diversity].

    PubMed

    Zhang, Jing; Zhang, Huiwen; Li, Xinyu; Su, Zhencheng; Zhang, Chenggang

    2006-06-01

    Soil microbes in terrestrial ecosystem carry out a series of important ecological functions, such as geo-chemical cycling of elements, degradation of pollutants, and buffering to the acute changes of environment, etc. Soil microbial ecological function has a close relation with soil function, and the changes in the structure and composition of soil microbial populations can directly affect the realization of soil function. Through their produced enzymes, soil microbes take part in a series of metabolic activities, and the functional genes of coded enzymes are the functional markers of microbes. In recent ten years, molecular ecology focusing on the functional gene diversity has been developed rapidly, which gives us a new cut-in point to understand soil microbial ecological function from the point of functional gene. This paper reviewed the research advances in the functional gene diversity correlated to soil microbial ecological function, with the perspectives in this field discussed.

  8. Metabolic Genes within Cyanophage Genomes: Implications for Diversity and Evolution

    PubMed Central

    Gao, E-Bin; Huang, Youhua; Ning, Degang

    2016-01-01

    Cyanophages, a group of viruses specifically infecting cyanobacteria, are genetically diverse and extensively abundant in water environments. As a result of selective pressure, cyanophages often acquire a range of metabolic genes from host genomes. The host-derived genes make a significant contribution to the ecological success of cyanophages. In this review, we summarize the host-derived metabolic genes, as well as their origin and roles in cyanophage evolution and important host metabolic pathways, such as the light-dependent reactions of photosynthesis, the pentose phosphate pathway, nutrient acquisition and nucleotide biosynthesis. We also discuss the suitability of the host-derived metabolic genes as potential diagnostic markers for the detection of genetic diversity of cyanophages in natural environments. PMID:27690109

  9. Species Accumulation Curves and Incidence-Based Species Richness Estimators to Appraise the Diversity of Cultivable Yeasts from Beech Forest Soils

    PubMed Central

    Yurkov, Andrey M.; Kemler, Martin; Begerow, Dominik

    2011-01-01

    Background Yeast-like fungi inhabit soils throughout all climatic zones in a great abundance. While recent estimations predicted a plethora of prokaryotic taxa in one gram of soil, similar data are lacking for fungi, especially yeasts. Methodology/Principal Findings We assessed the diversity of soil yeasts in different forests of central Germany using cultivation-based techniques with subsequent identification based on rDNA sequence data. Based on experiments using various pre-cultivation sample treatment and different cultivation media we obtained the highest number of yeasts by analysing mixed soil samples with a single nutrient-rich medium. Additionally, several species richness estimators were applied to incidence-based data of 165 samples. All of them predicted a similar range of yeast diversity, namely 14 to 16 species. Randomized species richness curves reached saturation in all applied estimators, thus indicating that the majority of species is detected after approximately 30 to 50 samples analysed. Conclusions/Significance In this study we demonstrate that robust species identification as well as mathematical approaches are essential to reliably estimate the sampling effort needed to describe soil yeast communities. This approach has great potential for optimisation of cultivation techniques and allows high throughput analysis in the future. PMID:21858201

  10. Expansion of Hexose Transporter Genes Was Associated with the Evolution of Aerobic Fermentation in Yeasts

    PubMed Central

    Lin, Zhenguo; Li, Wen-Hsiung

    2011-01-01

    The genetic basis of organisms’ adaptation to different environments is a central issue of molecular evolution. The budding yeast Saccharomyces cerevisiae and its relatives predominantly ferment glucose into ethanol even in the presence of oxygen. This was suggested to be an adaptation to glucose-rich habitats, but the underlying genetic basis of the evolution of aerobic fermentation remains unclear. In S. cerevisiae, the first step of glucose metabolism is transporting glucose across the plasma membrane, which is carried out by hexose transporter proteins. Although several studies have recognized that the rate of glucose uptake can affect how glucose is metabolized, the role of HXT genes in the evolution of aerobic fermentation has not been fully explored. In this study, we identified all members of the HXT gene family in 23 fully sequenced fungal genomes, reconstructed their evolutionary history to pinpoint gene gain and loss events, and evaluated their adaptive significance in the evolution of aerobic fermentation. We found that the HXT genes have been extensively amplified in the two fungal lineages that have independently evolved aerobic fermentation. In contrast, reduction of the number of HXT genes has occurred in aerobic respiratory species. Our study reveals a strong positive correlation between the copy number of HXT genes and the strength of aerobic fermentation, suggesting that HXT gene expansion has facilitated the evolution of aerobic fermentation. PMID:20660490

  11. Genomes, diversity and resistance gene analogues in Musa species.

    PubMed

    Azhar, M; Heslop-Harrison, J S

    2008-01-01

    Resistance genes (R genes) in plants are abundant and may represent more than 1% of all the genes. Their diversity is critical to the recognition and response to attack from diverse pathogens. Like many other crops, banana and plantain face attacks from potentially devastating fungal and bacterial diseases, increased by a combination of worldwide spread of pathogens, exploitation of a small number of varieties, new pathogen mutations, and the lack of effective, benign and cheap chemical control. The challenge for plant breeders is to identify and exploit genetic resistances to diseases, which is particularly difficult in banana and plantain where the valuable cultivars are sterile, parthenocarpic and mostly triploid so conventional genetic analysis and breeding is impossible. In this paper, we review the nature of R genes and the key motifs, particularly in the Nucleotide Binding Sites (NBS), Leucine Rich Repeat (LRR) gene class. We present data about identity, nature and evolutionary diversity of the NBS domains of Musa R genes in diploid wild species with the Musa acuminata (A), M. balbisiana (B), M. schizocarpa (S), M. textilis (T), M. velutina and M. ornata genomes, and from various cultivated hybrid and triploid accessions, using PCR primers to isolate the domains from genomic DNA. Of 135 new sequences, 75% of the sequenced clones had uninterrupted open reading frames (ORFs), and phylogenetic UPGMA tree construction showed four clusters, one from Musa ornata, one largely from the B and T genomes, one from A and M. velutina, and the largest with A, B, T and S genomes. Only genes of the coiled-coil (non-TIR) class were found, typical of the grasses and presumably monocotyledons. The analysis of R genes in cultivated banana and plantain, and their wild relatives, has implications for identification and selection of resistance genes within the genus which may be useful for plant selection and breeding and also for defining relationships and genome evolution

  12. Mobile Introns Shape the Genetic Diversity of Their Host Genes.

    PubMed

    Repar, Jelena; Warnecke, Tobias

    2017-04-01

    Self-splicing introns populate several highly conserved protein-coding genes in fungal and plant mitochondria. In fungi, many of these introns have retained their ability to spread to intron-free target sites, often assisted by intron-encoded endonucleases that initiate the homing process. Here, leveraging population genomic data from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Lachancea kluyveri, we expose nonrandom patterns of genetic diversity in exons that border self-splicing introns. In particular, we show that, in all three species, the density of single nucleotide polymorphisms increases as one approaches a mobile intron. Through multiple lines of evidence, we rule out relaxed purifying selection as the cause of uneven nucleotide diversity. Instead, our findings implicate intron mobility as a direct driver of host gene diversity. We discuss two mechanistic scenarios that are consistent with the data: either endonuclease activity and subsequent error-prone repair have left a mutational footprint on the insertion environment of mobile introns or nonrandom patterns of genetic diversity are caused by exonic coconversion, which occurs when introns spread to empty target sites via homologous recombination. Importantly, however, we show that exonic coconversion can only explain diversity gradients near intron-exon boundaries if the conversion template comes from outside the population. In other words, there must be pervasive and ongoing horizontal gene transfer of self-splicing introns into extant fungal populations. Copyright © 2017 Repar and Warnecke.

  13. C/N Ratio Drives Soil Actinobacterial Cellobiohydrolase Gene Diversity

    PubMed Central

    Prendergast-Miller, Miranda T.; Poonpatana, Pabhon; Farrell, Mark; Bissett, Andrew; Macdonald, Lynne M.; Toscas, Peter; Richardson, Alan E.; Thrall, Peter H.

    2015-01-01

    Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle. PMID:25710367

  14. Mobile Introns Shape the Genetic Diversity of Their Host Genes

    PubMed Central

    Repar, Jelena; Warnecke, Tobias

    2017-01-01

    Self-splicing introns populate several highly conserved protein-coding genes in fungal and plant mitochondria. In fungi, many of these introns have retained their ability to spread to intron-free target sites, often assisted by intron-encoded endonucleases that initiate the homing process. Here, leveraging population genomic data from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Lachancea kluyveri, we expose nonrandom patterns of genetic diversity in exons that border self-splicing introns. In particular, we show that, in all three species, the density of single nucleotide polymorphisms increases as one approaches a mobile intron. Through multiple lines of evidence, we rule out relaxed purifying selection as the cause of uneven nucleotide diversity. Instead, our findings implicate intron mobility as a direct driver of host gene diversity. We discuss two mechanistic scenarios that are consistent with the data: either endonuclease activity and subsequent error-prone repair have left a mutational footprint on the insertion environment of mobile introns or nonrandom patterns of genetic diversity are caused by exonic coconversion, which occurs when introns spread to empty target sites via homologous recombination. Importantly, however, we show that exonic coconversion can only explain diversity gradients near intron–exon boundaries if the conversion template comes from outside the population. In other words, there must be pervasive and ongoing horizontal gene transfer of self-splicing introns into extant fungal populations. PMID:28193728

  15. Allozyme gene diversities in some leaf beetles (Coleoptera: Chrysomelidae).

    PubMed

    Krafsur, E S

    1999-08-01

    Gene diversity at allozyme loci was investigated in the bean leaf beetle, Ceratoma trifurcata Forster; the elm leaf beetle, Xanthogaleruca luteola (Muller); the cottonwood leaf beetle, Chrysomela scripta Fabricus; the western corn rootworm, Diabrotica virgifera virgifera LeConte; the southern corn rootworm, also called the spotted cucumber beetle, D. undecimpunctata howardi Baker; the northern corn rootworm, D. barberi Smith and Lawrence; and the Colorado potato beetle, Leptinotarsa decemlineata (Say). Six of these species are economically important pests of crops and display adaptive traits that may correlate with genetic diversity. Gene diversity H(E) in bean leaf beetles was 17.7 +/- 4.0% among 32 loci. In western corn rootworms, H(E) = 4.8 +/- 2.0% among 36 loci, and in spotted cucumber beetles, H(E) = 11.9 +/- 2.7% among 39 loci. Diversity among 27 loci was 10.5 +/- 4.3% in the Colorado potato beetle. The data were compared with gene diversity estimates from other leaf beetle species in which heterozygosities varied from 0.3 to 21% and no correlation was detected among heterozygosities, geographic ranges, or population densities. Distributions of single-locus heterozygosities were consistent with selective neutrality of alleles.

  16. Yeast diversity in a traditional French cheese "Tomme d'orchies" reveals infrequent and frequent species with associated benefits.

    PubMed

    Ceugniez, Alexandre; Drider, Djamel; Jacques, Philippe; Coucheney, Françoise

    2015-12-01

    This study is aimed at unrevealing the yeast diversity of handmade cheese, Tomme d'orchies, produced and marketed in the north of France. A total of 185 yeast colonies were isolated from the surface and core of this cheese. From these, 80 morphologically different colonies were selected and subjected to rep-PCR analysis. The isolates were clustered into six distinct groups based on their DNA fingerprints. From each group, at least 30% of isolates were selected and identified to species level by biochemical characteristics (ID32C Api system) and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA regions. The isolates belonged to Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus, frequently isolated, and less frequently isolated Saturnispora mendoncae and Clavispora lusitaniae. Two isolates designated as Kluyveromyces lactis (isolate S-3-05) and Kluyveromyces marxianus (isolate S-2-05) were non-hemolytic, sensitive to antifungal compounds and able to inhibit the growth of pathogens including Candida albicans, Listeria monocytogenes and some bacilli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts.

    PubMed

    Varela, C; Kutyna, D R; Solomon, M R; Black, C A; Borneman, A; Henschke, P A; Pretorius, I S; Chambers, P J

    2012-09-01

    Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO(2). Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.

  18. Evaluation of Gene Modification Strategies for the Development of Low-Alcohol-Wine Yeasts

    PubMed Central

    Kutyna, D. R.; Solomon, M. R.; Black, C. A.; Borneman, A.; Henschke, P. A.; Pretorius, I. S.; Chambers, P. J.

    2012-01-01

    Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO2. Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites. PMID:22729542

  19. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    PubMed Central

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside. PMID:28134264

  20. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  1. Extraordinary diversity of visual opsin genes in dragonflies

    PubMed Central

    Futahashi, Ryo; Kawahara-Miki, Ryouka; Kinoshita, Michiyo; Yoshitake, Kazutoshi; Yajima, Shunsuke; Arikawa, Kentaro; Fukatsu, Takema

    2015-01-01

    Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15–33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies. PMID:25713365

  2. Extraordinary diversity of visual opsin genes in dragonflies.

    PubMed

    Futahashi, Ryo; Kawahara-Miki, Ryouka; Kinoshita, Michiyo; Yoshitake, Kazutoshi; Yajima, Shunsuke; Arikawa, Kentaro; Fukatsu, Takema

    2015-03-17

    Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.

  3. Improving freeze-tolerance of baker's yeast through seamless gene deletion of NTH1 and PUT1.

    PubMed

    Dong, Jian; Chen, Didi; Wang, Guanglu; Zhang, Cuiying; Du, Liping; Liu, Shanshan; Zhao, Yu; Xiao, Dongguang

    2016-06-01

    Baker's yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker's yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker's yeast using the method proposed in this paper.

  4. Yeast genes required for conversion of grape precursors to varietal thiols in wine.

    PubMed

    Santiago, Margarita; Gardner, Richard C

    2015-08-01

    Three varietal thiols are important for the tropical fruit aromas of Sauvignon blanc: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). These thiols are produced by yeast during fermentation from precursors in grape juice. Here we identify genes in Saccharomyces cerevisiae that are required for the transport and cleavage of two thiol precursors: cysteine-4MMP and glutathione-3MH. A full-length copy of IRC7 is absolutely required for the cleavage of both precursors in the tested strains; the deleted form of the enzyme found in most yeast strains is incapable of converting these compounds into detectable thiols. By using strains that overexpress full-length IRC7, we further show that the glutathione transporter OPT1 and the transpeptidase CIS2 are also required for conversion of glut-3MH to its varietal thiol. No transporter for cys-4MMP was identified: a strain deleted for all nine known cysteine transport genes was still capable of converting cys-4MMP to its varietal thiol, and was also able to take up cysteine at high concentrations. Based on these results, we conclude that cysteine and glutathione precursors make a relatively minor contribution to 3MH production from most grape juices. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Similar environments but diverse fates: Responses of budding yeast to nutrient deprivation

    PubMed Central

    Honigberg, Saul M.

    2016-01-01

    Diploid budding yeast (Saccharomyces cerevisiae) can adopt one of several alternative differentiation fates in response to nutrient limitation, and each of these fates provides distinct biological functions. When different strain backgrounds are taken into account, these various fates occur in response to similar environmental cues, are regulated by the same signal transduction pathways, and share many of the same master regulators. I propose that the relationships between fate choice, environmental cues and signaling pathways are not Boolean, but involve graded levels of signals, pathway activation and master-regulator activity. In the absence of large differences between environmental cues, small differences in the concentration of cues may be reinforced by cell-to-cell signals. These signals are particularly essential for fate determination within communities, such as colonies and biofilms, where fate choice varies dramatically from one region of the community to another. The lack of Boolean relationships between cues, signaling pathways, master regulators and cell fates may allow yeast communities to respond appropriately to the wide range of environments they encounter in nature. PMID:27917388

  6. A Mutator Affecting the Region of the Iso-1-Cytochrome c Gene in Yeast

    PubMed Central

    Liebman, Susan W.; Singh, Arjun; Sherman, Fred

    1979-01-01

    The mutator gene DEL1 in the yeast Saccharomyces cerevisiae causes a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYC1, which determines the structure of iso-1-cytochrome c; RAD7, which controls UV sensitivity; and OSM1, which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments.—The multisite mutations arise at a rate of approximately 10-5 to 10-6 per cell per division in DEL1 strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cyc1 mutants at a low rate of approximately 10-8 to 10-9. No evidence for multisite mutations was obtained upon analysis of numerous spontaneous ade1, ade2, met2 and met15 mutants isolated in a DEL1 strain. DEL1 segregates as a single Mendelian gene closely linked to the CYC1 locus. DEL1 appears to be both cis- and trans-dominant. The location of the DEL1 gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself. PMID:231539

  7. Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression.

    PubMed Central

    Clare, J; Farabaugh, P

    1985-01-01

    We have determined the DNA sequence of the transposable element Ty912 of yeast. The 5918-base-pair element encodes two genes, tya912 and tyb912, which specify proteins similar to sequence-specific DNA-binding proteins of Escherichia coli and retroviral reverse transcriptases, respectively. The tyb912 gene is atypical of eukaryotic genes since (i) it begins 1336 nucleotides into the Ty912 mRNA (i.e., downstream of the tya912 gene) and (ii) the first in-frame AUG is 921 nucleotides into the coding frame. Protein blot analysis of Ty-lacZ fusions shows that the tyb912 gene is translated starting at the 5' end of the tya912 gene and that the primary translational product is a tya912::tyb912 fusion protein. We have shown that synthesis of this fusion protein probably does not occur by RNA splicing. The data are consistent with a mechanism of translational frameshifting occurring within the region of overlap between the 3' end of tya912 and the 5' end of tyb912. Images PMID:2581255

  8. Effect of 21 different nitrogen sources on global gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Godard, Patrice; Urrestarazu, Antonio; Vissers, Stéphan; Kontos, Kevin; Bontempi, Gianluca; van Helden, Jacques; André, Bruno

    2007-04-01

    We compared the transcriptomes of Saccharomyces cerevisiae cells growing under steady-state conditions on 21 unique sources of nitrogen. We found 506 genes differentially regulated by nitrogen and estimated the activation degrees of all identified nitrogen-responding transcriptional controls according to the nitrogen source. One main group of nitrogenous compounds supports fast growth and a highly active nitrogen catabolite repression (NCR) control. Catabolism of these compounds typically yields carbon derivatives directly assimilable by a cell's metabolism. Another group of nitrogen compounds supports slower growth, is associated with excretion by cells of nonmetabolizable carbon compounds such as fusel oils, and is characterized by activation of the general control of amino acid biosynthesis (GAAC). Furthermore, NCR and GAAC appear interlinked, since expression of the GCN4 gene encoding the transcription factor that mediates GAAC is subject to NCR. We also observed that several transcriptional-regulation systems are active under a wider range of nitrogen supply conditions than anticipated. Other transcriptional-regulation systems acting on genes not involved in nitrogen metabolism, e.g., the pleiotropic-drug resistance and the unfolded-protein response systems, also respond to nitrogen. We have completed the lists of target genes of several nitrogen-sensitive regulons and have used sequence comparison tools to propose functions for about 20 orphan genes. Similar studies conducted for other nutrients should provide a more complete view of alternative metabolic pathways in yeast and contribute to the attribution of functions to many other orphan genes.

  9. Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae.

    PubMed Central

    Biggins, S; Bhalla, N; Chang, A; Smith, D L; Murray, A W

    2001-01-01

    Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor. PMID:11606525

  10. Diversity and abundance of phosphonate biosynthetic genes in nature

    PubMed Central

    Yu, Xiaomin; Doroghazi, James R.; Janga, Sarath C.; Zhang, Jun Kai; Circello, Benjamin; Griffin, Benjamin M.; Labeda, David P.; Metcalf, William W.

    2013-01-01

    Phosphonates, molecules containing direct carbon–phosphorus bonds, compose a structurally diverse class of natural products with interesting and useful biological properties. Although their synthesis in protozoa was discovered more than 50 y ago, the extent and diversity of phosphonate production in nature remains poorly characterized. The rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate, catalyzed by the enzyme PEP mutase (PepM), is shared by the vast majority of known phosphonate biosynthetic pathways. Thus, the pepM gene can be used as a molecular marker to examine the occurrence and abundance of phosphonate-producing organisms. Based on the presence of this gene, phosphonate biosynthesis is common in microbes, with ∼5% of sequenced bacterial genomes and 7% of genome equivalents in metagenomic datasets carrying pepM homologs. Similarly, we detected the pepM gene in ∼5% of random actinomycete isolates. The pepM-containing gene neighborhoods from 25 of these isolates were cloned, sequenced, and compared with those found in sequenced genomes. PEP mutase sequence conservation is strongly correlated with conservation of other nearby genes, suggesting that the diversity of phosphonate biosynthetic pathways can be predicted by examining PEP mutase diversity. We used this approach to estimate the range of phosphonate biosynthetic pathways in nature, revealing dozens of discrete groups in pepM amplicons from local soils, whereas hundreds were observed in metagenomic datasets. Collectively, our analyses show that phosphonate biosynthesis is both diverse and relatively common in nature, suggesting that the role of phosphonate molecules in the biosphere may be more important than is often recognized. PMID:24297932

  11. Gene diversity and genetic variation in lung flukes (genus Paragonimus).

    PubMed

    Blair, David; Nawa, Yukifumi; Mitreva, Makedonka; Doanh, Pham Ngoc

    2016-01-01

    Paragonimiasis caused by lung flukes (genus Paragonimus) is a neglected disease occurring in Asia, Africa and the Americas. The genus is species-rich, ancient and widespread. Genetic diversity is likely to be considerable, but investigation of this remains confined to a few populations of a few species. In recent years, studies of genetic diversity have moved from isoenzyme analysis to molecular phylogenetic analysis based on selected DNA sequences. The former offered better resolution of questions relating to allelic diversity and gene flow, whereas the latter is more suitable for questions relating to molecular taxonomy and phylogeny. A picture is emerging of a highly diverse taxon of parasites, with the greatest diversity found in eastern and southern Asia where ongoing speciation might be indicated by the presence of several species complexes. Diversity of lung flukes in Africa and the Americas is very poorly sampled. Functional molecules that might be of value for immunodiagnosis, or as targets for medical intervention, are of great interest. Characterisation of these from Paragonimus species has been ongoing for a number of years. However, the imminent release of genomic and transcriptomic data for several species of Paragonimus will dramatically increase the rate of discovery of such molecules, and illuminate their diversity within and between species. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Mutational specificity analysis: assay for mutations in the yeast SUP4-o gene.

    PubMed

    Kunz, Bernard A

    2014-01-01

    Mutational specificity analysis can yield valuable insights into processes that generate genetic change or maintain genetic stability. Powerful diagnostic tools for such analysis have been created by combining genetic assays for mutation with DNA sequencing. Here, steps for isolating spontaneous mutations in the yeast (Saccharomyces cerevisiae) suppressor tRNA gene SUP4-o as a prelude to sequence characterization are described (modifications of this protocol can be used to study induction of mutations by various physical or chemical agents). Mutations in SUP4-o are selected on drug-containing medium by virtue of their inactivation of suppressor activity. The small size, detailed knowledge of detectably mutable sites, and other features of the target gene facilitate subsequent analysis of these mutations.

  13. A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks

    PubMed Central

    Gubelmann, Carine; Waszak, Sebastian M; Isakova, Alina; Holcombe, Wiebke; Hens, Korneel; Iagovitina, Antonina; Feuz, Jean-Daniel; Raghav, Sunil K; Simicevic, Jovan; Deplancke, Bart

    2013-01-01

    The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein–DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF–DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers. PMID:23917988

  14. Detection of maltose fermentation genes in the baking yeast strains of Saccharomyces cerevisiae.

    PubMed

    Oda, Y; Tonomura, K

    1996-10-01

    The presence of any one of the five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 and MAL6) confers the ability to ferment maltose on the yeast Saccharomyces cerevisiae. Each locus is composed of three genes encoding maltose permease, alpha-glucosidase and MAL activator. Chromosomal DNA of seven representative baking strains has been separated by pulse-field gel electrophoresis and probed with three genes in MAL6 locus. The DNA bands to which all of the three MAL-derived probes simultaneously hybridized were chromosome VII carrying MAL1 in all of the strains tested, chromosome XI carrying MAL4 in six strains, chromosome III carrying MAL2 in three strains and chromosomes II and VIII carrying MAL3 and MAL6, respectively, in the one strain. The number of MAL loci in baking strains was comparable to those of brewing strains.

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

    PubMed

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

    2014-06-05

    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.

  16. Diverse Antibiotic Resistance Genes in Dairy Cow Manure

    PubMed Central

    Wichmann, Fabienne; Udikovic-Kolic, Nikolina; Andrew, Sheila; Handelsman, Jo

    2014-01-01

    ABSTRACT Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. PMID:24757214

  17. Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.

    PubMed

    Bakkali, F; Averbeck, S; Averbeck, D; Zhiri, A; Idaomar, M

    2005-08-01

    In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.

  18. Gene Catchr—Gene Cloning And Tagging for Caenorhabditis elegans using yeast Homologous Recombination: a novel approach for the analysis of gene expression

    PubMed Central

    Sassi, Holly E.; Renihan, Stephanie; Spence, Andrew M.; Cooperstock, Ramona L.

    2005-01-01

    Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression. PMID:16254074

  19. A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes.

    PubMed

    Kumar, Yatendra; Bhargava, Purnima

    2013-06-17

    RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at -140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5' NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin remodeler Isw1, aided by Isw2 and RSC

  20. Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation.

    PubMed

    Chiva, Rosana; López-Malo, Maria; Salvadó, Zoel; Mas, Albert; Guillamón, Jósé Manuel

    2012-11-01

    Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions.

  1. Rapid Expansion and Functional Divergence of Subtelomeric Gene Families in Yeasts

    PubMed Central

    Brown, Chris A.; Murray, Andrew W.; Verstrepen, Kevin J.

    2010-01-01

    Summary Background Subtelomeres, regions proximal to telomeres, exhibit characteristics unique to eukaryotic genomes. Genes residing in these loci are subject to epigenetic regulation and elevated rates of both meiotic and mitotic recombination. However, most genome sequences do not contain assembled subtelomeric sequences, and, as a result, subtelomeres are often overlooked in comparative genomics. Results We study the evolution and functional divergence of subtelomeric gene families in the yeast lineage. Our computational results show that subtelomeric families are evolving and expanding much faster than families that do not contain subtelomeric genes. Focusing on three related subtelomeric MAL gene families involved in disaccharide metabolism that show typical patterns of rapid expansion and evolution, we show experimentally how frequent duplication events followed by functional divergence yields novel alleles that allow metabolism of different carbohydrates. Conclusions Taken together, our computational and experimental analyses show that the extraordinary instability of eukaryotic subtelomeres supports rapid adaptation to novel niches by promoting gene recombination and duplication followed by functional divergence of the alleles. PMID:20471265

  2. Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity De Novo in the Yeast Cryptococcus neoformans

    PubMed Central

    Li, Wenjun; Floyd-Averette, Anna; Mieczkowski, Piotr; Dietrich, Fred S.; Heitman, Joseph

    2013-01-01

    Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments. PMID:24058295

  3. Diverse antibiotic resistance genes in dairy cow manure.

    PubMed

    Wichmann, Fabienne; Udikovic-Kolic, Nikolina; Andrew, Sheila; Handelsman, Jo

    2014-04-22

    Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected

  4. Detection, diversity and expression of aerobic bacterial arsenite oxidase genes.

    PubMed

    Inskeep, William P; Macur, Richard E; Hamamura, Natsuko; Warelow, Thomas P; Ward, Seamus A; Santini, Joanne M

    2007-04-01

    The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.

  5. Prioritization of gene regulatory interactions from large-scale modules in yeast

    PubMed Central

    Lee, Ho-Joon; Manke, Thomas; Bringas, Ricardo; Vingron, Martin

    2008-01-01

    Background The identification of groups of co-regulated genes and their transcription factors, called transcriptional modules, has been a focus of many studies about biological systems. While methods have been developed to derive numerous modules from genome-wide data, individual links between regulatory proteins and target genes still need experimental verification. In this work, we aim to prioritize regulator-target links within transcriptional modules based on three types of large-scale data sources. Results Starting with putative transcriptional modules from ChIP-chip data, we first derive modules in which target genes show both expression and function coherence. The most reliable regulatory links between transcription factors and target genes are established by identifying intersection of target genes in coherent modules for each enriched functional category. Using a combination of genome-wide yeast data in normal growth conditions and two different reference datasets, we show that our method predicts regulatory interactions with significantly higher predictive power than ChIP-chip binding data alone. A comparison with results from other studies highlights that our approach provides a reliable and complementary set of regulatory interactions. Based on our results, we can also identify functionally interacting target genes, for instance, a group of co-regulated proteins related to cell wall synthesis. Furthermore, we report novel conserved binding sites of a glycoprotein-encoding gene, CIS3, regulated by Swi6-Swi4 and Ndd1-Fkh2-Mcm1 complexes. Conclusion We provide a simple method to prioritize individual TF-gene interactions from large-scale transcriptional modules. In comparison with other published works, we predict a complementary set of regulatory interactions which yields a similar or higher prediction accuracy at the expense of sensitivity. Therefore, our method can serve as an alternative approach to prioritization for further experimental studies. PMID

  6. Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts.

    PubMed

    Gonçalves, Margarida; Pontes, Ana; Almeida, Pedro; Barbosa, Raquel; Serra, Marta; Libkind, Diego; Hutzler, Mathias; Gonçalves, Paula; Sampaio, José Paulo

    2016-10-24

    Beer is one of the oldest alcoholic beverages and is produced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S. cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single or multiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked to brewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequence of fundamental differences in the modes of beer and wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts.

    PubMed

    Fierro-Risco, Jesús; Rincón, Ana María; Benítez, Tahía; Codón, Antonio C

    2013-08-01

    Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.

  8. Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

    PubMed Central

    Kamenova, Ivanka; Warfield, Linda

    2014-01-01

    Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. PMID:24865972

  9. Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

    PubMed

    Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

    2014-08-01

    Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Copper and the ACE1 Regulatory Protein Reversibly Induce Yeast Metallothionein Gene Transcription in a Mouse Extract

    NASA Astrophysics Data System (ADS)

    Cizewski Culotta, Valeria; Hsu, Tsao; Hu, Stella; Furst, Peter; Hamer, Dean

    1989-11-01

    We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.

  11. Direct TFIIA-TFIID protein contacts drive budding yeast ribosomal protein gene transcription.

    PubMed

    Layer, Justin H; Weil, P Anthony

    2013-08-09

    We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo.

  12. Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription*

    PubMed Central

    Layer, Justin H.; Weil, P. Anthony

    2013-01-01

    We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

  13. Cdc7-Dbf4 is a gene-specific regulator of meiotic transcription in yeast.

    PubMed

    Lo, Hsiao-Chi; Kunz, Ryan C; Chen, Xiangyu; Marullo, Allison; Gygi, Steven P; Hollingsworth, Nancy M

    2012-01-01

    Meiosis divides the chromosome number of the cell in half by having two rounds of chromosome segregation follow a single round of chromosome duplication. The first meiotic division is unique in that homologous pairs of sister chromatids segregate to opposite poles. Recent work in budding and fission yeast has shown that the cell cycle kinase, Cdc7-Dbf4, is required for many meiosis-specific chromosomal functions necessary for proper disjunction at meiosis I. This work reveals another role for Cdc7 in meiosis as a gene-specific regulator of the global transcription factor, Ndt80, which is required for exit from pachytene and entry into the meiotic divisions in budding yeast. Cdc7-Dbf4 promotes NDT80 transcription by relieving repression mediated by a complex of Sum1, Rfm1, and a histone deacetylase, Hst1. Sum1 exhibits meiosis-specific Cdc7-dependent phosphorylation, and mass spectrometry analysis reveals a dynamic and complex pattern of phosphorylation events, including four constitutive cyclin-dependent kinase (Cdk1) sites and 11 meiosis-specific Cdc7-Dbf4-dependent sites. Analysis of various phosphorylation site mutants suggests that Cdc7 functions with both Cdk1 and the meiosis-specific kinase Ime2 to control this critical transition point during meiosis.

  14. Molecular phylogenetics of the genus Rhodotorula and related basidiomycetous yeasts inferred from the mitochondrial cytochrome b gene.

    PubMed

    Biswas, S K; Yokoyama, K; Nishimura, K; Miyaji, M

    2001-05-01

    Phylogenetic relationships of basidiomycetous yeasts, especially of the genus Rhodotorula, were studied using partial sequences of the mitochondrial cytochrome b gene. The results demonstrated that the basidiomycetous yeasts under investigation distributed into two main clusters: one containing Tremellales, Filobasidiales and their anamorphs and the other containing Ustilaginales, Sporidiales and their anamorphs. This clustering in turn correlates with cell wall biochemistry, presence or absence of xylose, and septal ultrastructure, dolipore or simple pore. Bullera, Bulleromyces, Filobasidiella, Cryptococcus and Trichosporon, yeasts of the former cluster, contain xylose in the cell wall and have dolipore septa. In contrast yeasts of the latter cluster, which included Bensingtonia, Erythrobasidium, Leucosporidium, Malassezia, Rhodosporidium, Rhodotorula, Sporidiobolus, Sporobolomyces and Ustilago, have no xylose in the cell wall and have a simple pore septum. Yeasts of the latter group could be further divided into four clades (A-D). Species of Rhodotorula were distributed in all of these clades, indicating the polyphyletic nature of the genus. A limited number of Rhodotorula species demonstrated identical sequences, for example Rhodotorula bacarum and Rhodotorula foliorum, Rhodotorula fujisanensis and Rhodotorula futronensis, Rhodotorula glutinis var. dairenensis and Rhodotorula mucilaginosa. However, all the other test species of the genus Rhodotorula were well separated based on their 396 bp nucleotide sequences. These results demonstrate the effectiveness of the use of cytochrome b sequences for both species identification and the study of phylogenetic relationships among basidiomycetous yeasts.

  15. Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (Sarcophilus harrisii).

    PubMed

    Morris, Katrina M; Wright, Belinda; Grueber, Catherine E; Hogg, Carolyn; Belov, Katherine

    2015-08-01

    The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll-like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome-level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole-genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29-220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long-term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad-scale immunogenetic diversity analysis in threatened species. © 2015 The Authors. Molecular Ecology Published

  16. Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

    PubMed

    Tongaonkar, Prasad; French, Sarah L; Oakes, Melanie L; Vu, Loan; Schneider, David A; Beyer, Ann L; Nomura, Masayasu

    2005-07-19

    Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

  17. Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

    PubMed

    Brown, A J; Planta, R J; Restuhadi, F; Bailey, D A; Butler, P R; Cadahia, J L; Cerdan, M E; De Jonge, M; Gardner, D C; Gent, M E; Hayes, A; Kolen, C P; Lombardia, L J; Murad, A M; Oliver, R A; Sefton, M; Thevelein, J M; Tournu, H; van Delft, Y J; Verbart, D J; Winderickx, J; Oliver, S G

    2001-06-15

    The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

  18. Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations

    PubMed Central

    Brown, Alistair J.P.; Planta, Rudi J.; Restuhadi, Fajar; Bailey, David A.; Butler, Philip R.; Cadahia, Jose L.; Cerdan, M.Esperanza; De Jonge, Martine; Gardner, David C.J.; Gent, Manda E.; Hayes, Andrew; Kolen, Carin P.A.M.; Lombardia, Luis J.; Murad, Abdul Munir Abdul; Oliver, Rachel A.; Sefton, Mark; Thevelein, Johan M.; Tournu, Helene; van Delft, Yvon J.; Verbart, Dennis J.; Winderickx, Joris; Oliver, Stephen G.

    2001-01-01

    The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs. PMID:11406594

  19. Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans.

    PubMed

    Böer, Erik; Schröter, Anja; Bode, Rüdiger; Piontek, Michael; Kunze, Gotthard

    2009-02-01

    In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single-copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO(3). We quantitatively analysed the promoter activities by qRT-PCR and after fusing individual promoter fragments to the phytase (phyK) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO(3) are compatible with these results. Both enzymes show optimal activity at around 42 degrees C and near-neutral pH, and require FAD as a co-factor and NADPH as electron donor.

  20. Dissecting Dynamic Genetic Variation That Controls Temporal Gene Response in Yeast

    PubMed Central

    Brodt, Avital; Botzman, Maya; David, Eyal; Gat-Viks, Irit

    2014-01-01

    Inter-individual variation in regulatory circuits controlling gene expression is a powerful source of functional information. The study of associations among genetic variants and gene expression provides important insights about cell circuitry but cannot specify whether and when potential variants dynamically alter their genetic effect during the course of response. Here we develop a computational procedure that captures temporal changes in genetic effects, and apply it to analyze transcription during inhibition of the TOR signaling pathway in segregating yeast cells. We found a high-order coordination of gene modules: sets of genes co-associated with the same genetic variant and sharing a common temporal genetic effect pattern. The temporal genetic effects of some modules represented a single state-transitioning pattern; for example, at 10–30 minutes following stimulation, genetic effects in the phosphate utilization module attained a characteristic transition to a new steady state. In contrast, another module showed an impulse pattern of genetic effects; for example, in the poor nitrogen sources utilization module, a spike up of a genetic effect at 10–20 minutes following stimulation reflected inter-individual variation in the timing (rather than magnitude) of response. Our analysis suggests that the same mechanism typically leads to both inter-individual variation and the temporal genetic effect pattern in a module. Our methodology provides a quantitative genetic approach to studying the molecular mechanisms that shape dynamic changes in transcriptional responses. PMID:25474467

  1. Characterization of global gene expression during assurance of lifespan extension by caloric restriction in budding yeast.

    PubMed

    Choi, Kyung-Mi; Kwon, Young-Yon; Lee, Cheol-Koo

    2013-12-01

    Caloric restriction (CR) is the best-studied intervention known to delay aging and extend lifespan in evolutionarily distant organisms ranging from yeast to mammals in the laboratory. Although the effect of CR on lifespan extension has been investigated for nearly 80years, the molecular mechanisms of CR are still elusive. Consequently, it is important to understand the fundamental mechanisms of when and how lifespan is affected by CR. In this study, we first identified the time-windows during which CR assured cellular longevity by switching cells from culture media containing 2% or 0.5% glucose to water, which allows us to observe CR and non-calorically-restricted cells under the same conditions. We also constructed time-dependent gene expression profiles and selected 646 genes that showed significant changes and correlations with the lifespan-extending effect of CR. The positively correlated genes participated in transcriptional regulation, ribosomal RNA processing and nuclear genome stability, while the negatively correlated genes were involved in the regulation of several metabolic pathways, endoplasmic reticulum function, stress response and cell cycle progression. Furthermore, we discovered major upstream regulators of those significantly changed genes, including AZF1 (YOR113W), HSF1 (YGL073W) and XBP1 (YIL101C). Deletions of two genes, AZF1 and XBP1 (HSF1 is essential and was thus not tested), were confirmed to lessen the lifespan extension mediated by CR. The absence of these genes in the tor1Δ and ras2Δ backgrounds did show non-overlapping effects with regard to CLS, suggesting differences between the CR mechanism for Tor and Ras signaling. © 2013.

  2. Four mating-type genes control sexual differentiation in the fission yeast.

    PubMed

    Kelly, M; Burke, J; Smith, M; Klar, A; Beach, D

    1988-05-01

    The mating-type region of fission yeast consists of three components, mat1, mat2-P and mat3-M, each separated by 15 kb. Cell-type is determined by the alternate allele present at mat1, either P in an h+ or M in an h- cell. mat2-P and mat3-M serve as donors of information that is transposed to mat1 during a switch of mating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating-type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at mat2-P and mat3-M but absent at mat1 (H3; 57 bp), and may be involved in transcriptional repression of these cassettes. mat1-P and mat1-M each encode two genes (Pc; 118 amino acids, Pi; 159 amino acids, Mc; 181 amino acids and Mi; 42 amino acids). Introduction of opal or frame-shift mutations into the open-reading-frame of each gene revealed that Pc and Mc are necessary and sufficient for mating and confer an h+ or h- mating type respectively. All four genes are required for meiotic competence in an h+/h- diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen-free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.

  3. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast.

    PubMed

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y; Brem, Rachel B

    2016-06-27

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1 Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1 Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them.

  4. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast

    PubMed Central

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y.; Brem, Rachel B.

    2016-01-01

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1. Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1. Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them. PMID:27190003

  5. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication.

    PubMed

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  6. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    PubMed Central

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  7. Yeast community survey in the Tagus estuary.

    PubMed

    de Almeida, João M G C F

    2005-07-01

    The yeast community in the waters of the Tagus estuary, Portugal, was followed for over a year in order to assess its dynamics. Yeast occurrence and incidence were measured and this information was related to relevant environmental data. Yeast occurrence did not seem to depend upon tides, but river discharge had a dramatic impact both on the density and diversity of the community. The occurrence of some yeasts was partially correlated with faecal pollution indicators. Yeast isolates were characterized by microsatellite primed PCR (MSP-PCR) fingerprinting and rRNA gene sequencing. The principal species found were Candida catenulata, C. intermedia, C. parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Pichia guilliermondii, Rhodotorula mucilaginosa and Rhodosporidium diobovatum. The incidence of these species was evaluated against the environmental context of the samples and the current knowledge about the substrates from which they are usually isolated.

  8. Population structure and comparative genome hybridization of European flor yeast reveal a unique group of Saccharomyces cerevisiae strains with few gene duplications in their genome.

    PubMed

    Legras, Jean-Luc; Erny, Claude; Charpentier, Claudine

    2014-01-01

    Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation.

  9. Population Structure and Comparative Genome Hybridization of European Flor Yeast Reveal a Unique Group of Saccharomyces cerevisiae Strains with Few Gene Duplications in Their Genome

    PubMed Central

    Legras, Jean-Luc; Erny, Claude; Charpentier, Claudine

    2014-01-01

    Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation. PMID:25272156

  10. Genetic diversity in the feline leukemia virus gag gene.

    PubMed

    Kawamura, Maki; Watanabe, Shinya; Odahara, Yuka; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

    2015-06-02

    Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    SciTech Connect

    Shih, Patrick M.; Vuu, Khanh; Mansoori, Nasim; Ayad, Leïla; Louie, Katherine B.; Bowen, Benjamin P.; Northen, Trent R.; Loqué, Dominique

    2016-10-26

    The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. But, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. We describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We also demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies for stacking genes and traits to address many impending environmental and agricultural challenges.

  12. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    PubMed Central

    Shih, Patrick M.; Vuu, Khanh; Mansoori, Nasim; Ayad, Leïla; Louie, Katherine B.; Bowen, Benjamin P.; Northen, Trent R.; Loqué, Dominique

    2016-01-01

    The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. However, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. Here, we describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies for stacking genes and traits to address many impending environmental and agricultural challenges. PMID:27782150

  13. [RAD18 gene product of yeast Saccharomyces cerevisiae controls mutagenesis induced by hydrogen peroxide].

    PubMed

    Kozhina, T N; Korolev, V G

    2012-04-01

    Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6-Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.

  14. Cloning of the transketolase gene from erythritol-producing yeast Candida magnoliae.

    PubMed

    Yoo, Boung-Hyuk; Park, Eun-Hee; Seo, Jin-Ho; Kim, Myoung-Dong

    2014-10-01

    The entire nucleotide sequence of the TKL1 gene encoding transketolase (TKL) in an erythritolproducing yeast of Candida magnoliae was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed an open reading frame of C. magnoliae TKL1 (CmTKL1) that spans 2,088 bp and encodes 696 amino acids, sharing 61.7% amino acid identity to Kluyveromyces lactis TKL. Functional analysis showed that CmTKL1 complemented a Saccharomyces cerevisiae tkl1 tkl2 double mutant for growth in the absence of aromatic amino acids and restored transketolase activity in this mutant. An enzyme activity assay and RT-PCR revealed that the expression of CmTKL1 is induced by fructose, H2O2, and KCl. The GenBank accession number for C. magnoliae TKL1 is KF751756.

  15. A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    DOE PAGES

    Shih, Patrick M.; Vuu, Khanh; Mansoori, Nasim; ...

    2016-10-26

    The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. But, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. We describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We also demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies formore » stacking genes and traits to address many impending environmental and agricultural challenges.« less

  16. Localization of genes for the double-stranded RNA killer virus of yeast.

    PubMed Central

    Welsh, J D; Leibowitz, M J

    1982-01-01

    The M double-stranded RNA (ds RNA) genome segment of the cytoplasmically inherited killer virus of yeast codes for two polypeptides when denatured and translated in vitro: a previously known 32,000-dalton peptide and a newly discovered 19,000-dalton peptide (NaDodSO4/polyacrylamide gel electrophoresis). An internal 190-base-pair region of the ds RNA is selectively degraded by S1 nuclease treatment at 65 degrees C, resulting in two ds RNA fragments which contain the termini of the original ds RNA. The larger fragment codes for the 32,000-dalton polypeptide and the smaller fragment codes for the 19,000-dalton polypeptide. Thus, the two gene products of M are encoded by distinct regions of this ds RNA. Images PMID:7038685

  17. Isolation and Characterization of a Gene Specific to Lager Brewing Yeast That Encodes a Branched-Chain Amino Acid Permease

    PubMed Central

    Kodama, Yukiko; Omura, Fumihiko; Ashikari, Toshihiko

    2001-01-01

    We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts. PMID:11472919

  18. A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes.

    PubMed

    Esser, Christian; Ahmadinejad, Nahal; Wiegand, Christian; Rotte, Carmen; Sebastiani, Federico; Gelius-Dietrich, Gabriel; Henze, Katrin; Kretschmann, Ernst; Richly, Erik; Leister, Dario; Bryant, David; Steel, Michael A; Lockhart, Peter J; Penny, David; Martin, William

    2004-09-01

    Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.

  19. Improved fermentation performance of a lager yeast after repair of its AGT1 maltose and maltotriose transporter genes.

    PubMed

    Vidgren, Virve; Huuskonen, Anne; Virtanen, Hannele; Ruohonen, Laura; Londesborough, John

    2009-04-01

    The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous alpha-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24 degrees Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.

  20. Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae.

    PubMed

    Shima, Jun; Sakata-Tsuda, Yuko; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Kawamoto, Shinichi; Takano, Hiroyuki

    2003-01-01

    The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.

  1. Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production

    Treesearch

    Nian-Qing. Shi; Brian. Davis; Fred. Sherman; Jose. Cruz; Thomas W. Jeffries

    1999-01-01

    The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization...

  2. Machine learning techniques to identify putative genes involved in nitrogen catabolite repression in the yeast Saccharomyces cerevisiae

    PubMed Central

    Kontos, Kevin; Godard, Patrice; André, Bruno; van Helden, Jacques; Bontempi, Gianluca

    2008-01-01

    Background Nitrogen is an essential nutrient for all life forms. Like most unicellular organisms, the yeast Saccharomyces cerevisiae transports and catabolizes good nitrogen sources in preference to poor ones. Nitrogen catabolite repression (NCR) refers to this selection mechanism. All known nitrogen catabolite pathways are regulated by four regulators. The ultimate goal is to infer the complete nitrogen catabolite pathways. Bioinformatics approaches offer the possibility to identify putative NCR genes and to discard uninteresting genes. Results We present a machine learning approach where the identification of putative NCR genes in the yeast Saccharomyces cerevisiae is formulated as a supervised two-class classification problem. Classifiers predict whether genes are NCR-sensitive or not from a large number of variables related to the GATA motif in the upstream non-coding sequences of the genes. The positive and negative training sets are composed of annotated NCR genes and manually-selected genes known to be insensitive to NCR, respectively. Different classifiers and variable selection methods are compared. We show that all classifiers make significant and biologically valid predictions by comparing these predictions to annotated and putative NCR genes, and by performing several negative controls. In particular, the inferred NCR genes significantly overlap with putative NCR genes identified in three genome-wide experimental and bioinformatics studies. Conclusion These results suggest that our approach can successfully identify potential NCR genes. Hence, the dimensionality of the problem of identifying all genes involved in NCR is drastically reduced. PMID:19091052

  3. Effect of inhibition of the bc1 complex on gene expression profile in yeast.

    PubMed

    Bourges, Ingrid; Horan, Susannah; Meunier, Brigitte

    2005-08-19

    Because the respiratory chain is the major site of oxidation of the reduced equivalents and of energy production in aerobic cells, its inhibition has severe impact on the cells. Communication pathways from the respiratory chain are required to allow the cell to sense the defect and respond to it. In this work, we studied changes in gene expression induced by the treatment of yeast cells with myxothiazol, an inhibitor of the bc(1) complex, an enzyme of the respiratory chain. The pattern and time-course expression of the genes resemble those of the environmental stress response, a common gene expression program induced by sudden changes in the environment. In addition, the changes were, for most of the genes, mediated through the transcription factors Msn2/4, which play a central role in the cellular response to these stresses. By using a mutant with a myxothiazol-resistant bc(1) complex, we showed that the changes of expression of the majority of the genes was caused by the inhibition of the bc(1) complex but that other stresses might be involved. The expression pattern of CTT1, coding for a cytoplasmic catalase, was further studied. The expression of this gene was largely dependent on Msn2/4 and the inhibition of the cytochrome bc(1). Addition of oxidants of NADH was found to decrease the expression of CTT1 induced by myxothiazol treatment, suggesting that the accumulation of NADH caused by the inhibition of the respiratory chain may be involved in the signaling pathway from the mitochondria to the transcription factor.

  4. Generation of Diversity in Streptococcus mutans Genes Demonstrated by MLST

    PubMed Central

    Do, Thuy; Gilbert, Steven C.; Clark, Douglas; Ali, Farida; Fatturi Parolo, Clarissa C.; Maltz, Marisa; Russell, Roy R.; Holbrook, Peter; Wade, William G.; Beighton, David

    2010-01-01

    Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However π, nucleotide diversity per site, was low for all loci being in the range 0.019–0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3–1.15] compared to 8.3 [95% confidence interval 5.0–14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra

  5. Diverse alkane hydroxylase genes in microorganisms and environments

    PubMed Central

    Nie, Yong; Chi, Chang-Qiao; Fang, Hui; Liang, Jie-Liang; Lu, She-Lian; Lai, Guo-Li; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-01-01

    AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature. PMID:24829093

  6. Activation of the cell integrity pathway is channelled through diverse signalling elements in fission yeast.

    PubMed

    Barba, Gregorio; Soto, Teresa; Madrid, Marisa; Núñez, Andrés; Vicente, Jeronima; Gacto, Mariano; Cansado, José

    2008-04-01

    MAPK Pmk1p is the central element of a cascade involved in the maintenance of cell integrity and other functions in Schizosaccharomyces pombe. Pmk1p becomes activated by multiple stressing situations and also during cell separation. GTPase Rho2p acts upstream of the protein kinase C homolog Pck2p to activate the Pmk1 signalling pathway through direct interaction with MAPKKK Mkh1p. In this work we analyzed the functional significance of both Rho2p and Pck2p in the transduction of various stress signals by the cell integrity pathway. The results indicate that basal Pmk1p activity can be positively regulated by alternative mechanisms which are independent on the control by Rho2p and/or Pck2p. Unexpectedly, Pck1p, another protein kinase C homolog, negatively modulates Pmk1p basal activity by an unknown mechanism. Moreover, different elements appear to regulate the stress-induced activation of Pmk1p depending on the nature of the triggering stimuli. Whereas Pmk1p activation induced by hyper- or hypotonic stresses is channeled through Rho2p-Pck2p, other stressors, like glucose deprivation or cell wall disturbance, are transduced via other pathways in addition to that of Rho2p-Pck2p. On the contrary, Pmk1p activation observed during cell separation or after treatment with hydrogen peroxide does not involve Rho2p-Pck2p. Finally, Pck2p function is critical to maintain a Pmk1p basal activity that allows Pmk1p activation induced by heat stress. These data demonstrate the existence of a complex signalling network modulating Pmk1p activation in response to a variety of stresses in fission yeast.

  7. The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

    PubMed

    Flores Berrios, E P; Alba González, J F; Arrizon Gaviño, J P; Romano, P; Capece, A; Gschaedler Mathis, A

    2005-01-01

    The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

  8. Construction of a yeast artifical chromosome contig spanning the spinal muscular atrophy disease gene region

    SciTech Connect

    Kleyn, P.W.; Wang, C.H.; Vitale, E.; Pan, J.; Ross, B.M.; Grunn, A.; Palmer, D.A.; Warburton, D.; Brzustowicz, L.M.; Gilliam, T.G. ); Lien, L.L.; Kunkel, L.M. )

    1993-07-15

    The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene. 26 refs., 3 figs., 1 tab.

  9. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

    PubMed Central

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S.; Boeke, Jef D.

    2015-01-01

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer’s yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational “safeguard” control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10−10), consistent with their orthogonal nature and the individual escape frequencies of <10−6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance. PMID:25624482

  10. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

    PubMed Central

    Zeevi, Danny; Sharon, Eilon; Lotan-Pompan, Maya; Lubling, Yaniv; Shipony, Zohar; Raveh-Sadka, Tali; Keren, Leeat; Levo, Michal; Weinberger, Adina; Segal, Eran

    2011-01-01

    Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded. PMID:22009988

  11. Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region.

    PubMed Central

    Kleyn, P W; Wang, C H; Lien, L L; Vitale, E; Pan, J; Ross, B M; Grunn, A; Palmer, D A; Warburton, D; Brzustowicz, L M

    1993-01-01

    The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene. Images Fig. 1 PMID:8341701

  12. Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast

    PubMed Central

    North, Matthew; Tandon, Vickram J.; Thomas, Reuben; Loguinov, Alex; Gerlovina, Inna; Hubbard, Alan E.; Zhang, Luoping; Smith, Martyn T.; Vulpe, Chris D.

    2011-01-01

    Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease. PMID:21912624

  13. Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes.

    PubMed

    Cai, Yizhi; Agmon, Neta; Choi, Woo Jin; Ubide, Alba; Stracquadanio, Giovanni; Caravelli, Katrina; Hao, Haiping; Bader, Joel S; Boeke, Jef D

    2015-02-10

    Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.

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

    PubMed Central

    Andrade, Rosângela V; Paes, Hugo C; Nicola, André M; de Carvalho, Maria José A; Fachin, Ana Lúcia; Cardoso, Renato S; Silva, Simoneide S; Fernandes, Larissa; Silva, Silvana P; Donadi, Eduardo A; Sakamoto-Hojo, Elza T; Passos, Geraldo AS; Soares, Célia MA; Brígido, Marcelo M; Felipe, Maria Sueli S

    2006-01-01

    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 together, these data

  15. Identification of Targetable FGFR Gene Fusions in Diverse Cancers

    PubMed Central

    Wu, Yi-Mi; Su, Fengyun; Kalyana-Sundaram, Shanker; Khazanov, Nick; Ateeq, Bushra; Cao, Xuhong; Lonigro, Robert J.; Vats, Pankaj; Wang, Rui; Lin, Su-Fang; Cheng, Ann-Joy; Kunju, Lakshmi P.; Siddiqui, Javed; Tomlins, Scott A.; Wyngaard, Peter; Sadis, Seth; Roychowdhury, Sameek; Hussain, Maha H.; Feng, Felix Y.; Zalupski, Mark M.; Talpaz, Moshe; Pienta, Kenneth J.; Rhodes, Daniel R.; Robinson, Dan R.; Chinnaiyan, Arul M.

    2013-01-01

    Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2 including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR gene fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Due to the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts which incorporate transcriptome analysis for gene fusions are poised to identify rare, targetable FGFR fusions across diverse cancer types. PMID:23558953

  16. Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

    PubMed Central

    2012-01-01

    Background Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides

  17. In-silico identification and characterization of organic and inorganic chemical stress responding genes in yeast (Saccharomyces cerevisiae).

    PubMed

    Barozai, Muhammad Younas Khan; Bashir, Farrukh; Muzaffar, Shafia; Afzal, Saba; Behlil, Farida; Khan, Muzaffar

    2014-10-15

    To study the life processes of all eukaryotes, yeast (Saccharomyces cerevisiae) is a significant model organism. It is also one of the best models to study the responses of genes at transcriptional level. In a living organism, gene expression is changed by chemical stresses. The genes that give response to chemical stresses will provide good source for the strategies in engineering and formulating mechanisms which are chemical stress resistant in the eukaryotic organisms. The data available through microarray under the chemical stresses like lithium chloride, lactic acid, weak organic acids and tomatidine were studied by using computational tools. Out of 9335 yeast genes, 388 chemical stress responding genes were identified and characterized under different chemical stresses. Some of these are: Enolases 1 and 2, heat shock protein-82, Yeast Elongation Factor 3, Beta Glucanase Protein, Histone H2A1 and Histone H2A2 Proteins, Benign Prostatic Hyperplasia, ras GTPase activating protein, Establishes Silent Chromatin protein, Mei5 Protein, Nondisjunction Protein and Specific Mitogen Activated Protein Kinase. Characterization of these genes was also made on the basis of their molecular functions, biological processes and cellular components. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes.

    PubMed

    Eldarov, M A; Baranov, M V; Dumina, M V; Shgun, A A; Andreeva, N A; Trilisenko, L V; Kulakovskaya, T V; Ryasanova, L P; Kulaev, I S

    2013-08-01

    The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.

  19. Draft genome sequence and transcriptome analysis of the wine spoilage yeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism and survival.

    PubMed

    Valdes, Jorge; Tapia, Paz; Cepeda, Victoria; Varela, Javier; Godoy, Liliana; Cubillos, Francisco A; Silva, Evelyn; Martinez, Claudio; Ganga, Maria Angélica

    2014-12-01

    Dekkera bruxellensis is the major contaminant yeast in the wine industry worldwide. Here, we present the draft genome sequence of D. bruxellensis LAMAP2480 isolated from a Chilean wine. Genomic evidence reveals shared and exclusive genes potentially involved in colonization and survival during alcoholic fermentation.

  20. Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Janowicz, Z A; Eckart, M R; Drewke, C; Roggenkamp, R O; Hollenberg, C P; Maat, J; Ledeboer, A M; Visser, C; Verrips, C T

    1985-05-10

    A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.

  1. Stress-tolerance of baker's-yeast (Saccharomyces cerevisiae) cells: stress-protective molecules and genes involved in stress tolerance.

    PubMed

    Shima, Jun; Takagi, Hiroshi

    2009-05-29

    During the fermentation of dough and the production of baker's yeast (Saccharomyces cerevisiae), cells are exposed to numerous environmental stresses (baking-associated stresses) such as freeze-thaw, high sugar concentrations, air-drying and oxidative stresses. Cellular macromolecules, including proteins, nucleic acids and membranes, are seriously damaged under stress conditions, leading to the inhibition of cell growth, cell viability and fermentation. To avoid lethal damage, yeast cells need to acquire a variety of stress-tolerant mechanisms, for example the induction of stress proteins, the accumulation of stress protectants, changes in membrane composition and repression of translation, and by regulating the corresponding gene expression via stress-triggered signal-transduction pathways. Trehalose and proline are considered to be critical stress protectants, as is glycerol. It is known that these molecules are effective for providing protection against various types of environmental stresses. Modifications of the metabolic pathways of trehalose and proline by self-cloning methods have significantly increased tolerance to baking-associated stresses. To clarify which genes are required for stress tolerance, both a comprehensive phenomics analysis and a functional genomics analysis were carried out under stress conditions that simulated those occurring during the commercial baking process. These analyses indicated that many genes are involved in stress tolerance in yeast. In particular, it was suggested that vacuolar H+-ATPase plays important roles in yeast cells under stress conditions.

  2. GLK gene pairs regulate chloroplast development in diverse plant species.

    PubMed

    Fitter, David W; Martin, David J; Copley, Martin J; Scotland, Robert W; Langdale, Jane A

    2002-09-01

    Chloroplast biogenesis is a complex process that requires close co-ordination between two genomes. Many of the proteins that accumulate in the chloroplast are encoded by the nuclear genome, and the developmental transition from proplastid to chloroplast is regulated by nuclear genes. Here we show that a pair of Golden 2-like (GLK) genes regulates chloroplast development in Arabidopsis. The GLK proteins are members of the GARP superfamily of transcription factors, and phylogenetic analysis demonstrates that the maize, rice and Arabidopsis GLK gene pairs comprise a distinct group within the GARP superfamily. Further phylogenetic analysis suggests that the gene pairs arose through separate duplication events in the monocot and dicot lineages. As in rice, AtGLK1 and AtGLK2 are expressed in partially overlapping domains in photosynthetic tissue. Insertion mutants demonstrate that this expression pattern reflects a degree of functional redundancy as single mutants display normal phenotypes in most photosynthetic tissues. However, double mutants are pale green in all photosynthetic tissues and chloroplasts exhibit a reduction in granal thylakoids. Products of several genes involved in light harvesting also accumulate at reduced levels in double mutant chloroplasts. GLK genes therefore regulate chloroplast development in diverse plant species.

  3. Sequence diversity of mating-type genes in Phaeosphaeria avenaria.

    PubMed

    Ueng, Peter P; Dai, Qun; Cui, Kai-rong; Czembor, Paweł C; Cunfer, Barry M; Tsang, H; Arseniuk, Edward; Bergstrom, Gary C

    2003-05-01

    Phaeosphaeria avenaria, one of the causal agents of stagonospora leaf blotch diseases in cereals, is composed of two subspecies, P. avenaria f. sp. triticea (Pat) and P. avenaria f. sp. avenaria (Paa). The Pat subspecies was grouped into Pat1-Pat3, based on restriction fragment length polymorphism (RFLP) and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences in previous studies. Mating-type genes and their potential use in phylogeny and molecular classification were studied by DNA hybridization and PCR amplification. The majority of Pat1 isolates reported to be homothallic and producing sexual reproduction structures on cultural media had only the MAT1-1 gene. Minor sequence variations were found in the conserved region of MAT1-1 gene in Pat1 isolates. However, both mating-type genes, MAT1-1 and MAT1-2, were identified in P. avenaria isolates represented by ATCC12277 from oats (Paa) and the Pat2 isolates from foxtail barley ( Hordeum jubatum L.). Cluster analyses based on mating-type gene conserved regions revealed that cereal Phaeosphaeria is not phylogenetically closely related to other ascomycetes, including Mycosphaerella graminicola (anamorph Septoria tritici). The sequence diversity of mating-type genes in Pat and Paa supports our previous phylogenetic relationship and molecular classification based on RFLP fingerprinting and rDNA ITS sequences.

  4. The Yeast Bsd2-1 Mutation Influences Both the Requirement for Phosphatidylinositol Transfer Protein Function and Derepression of Phospholipid Biosynthetic Gene Expression in Yeast

    PubMed Central

    Kagiwada, S.; Kearns, B. G.; McGee, T. P.; Fang, M.; Hosaka, K.; Bankaitis, V. A.

    1996-01-01

    The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino(-) phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the ``bypass Sec14p'' phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidylinositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed. PMID:8725219

  5. Isolation and Diversity Analysis of Resistance Gene Homologues from Switchgrass

    PubMed Central

    Zhu, Qihui; Bennetzen, Jeffrey L.; Smith, Shavannor M.

    2013-01-01

    Resistance gene homologs (RGHs) were isolated from the switchgrass variety Alamo by a combination of polymerase chain reaction and expressed sequence tag (EST) database mining. Fifty-eight RGHs were isolated by polymerase chain reaction and 295 RGHs were identified in 424,545 switchgrass ESTs. Four nucleotide binding site−leucine-rich repeat RGHs were selected to investigate RGH haplotypic diversity in seven switchgrass varieties chosen for their representation of a broad range of the switchgrass germplasm. Lowland and upland ecotypes were found to be less similar, even from nearby populations, than were more distant populations with similar growth environments. Most (83.5%) of the variability in these four RGHs was found to be attributable to the within-population component. The difference in nucleotide diversity between and within populations was observed to be small, whereas this diversity is maintained to similar degrees at both population and ecotype levels. The results also revealed that the analyzed RGHs were under positive selection in the studied switchgrass accessions. Intragenic recombination was detected in switchgrass RGHs, thereby demonstrating an active genetic process that has the potential to generate new resistance genes with new specificities that might act against newly-arising pathogen races. PMID:23589518

  6. A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis.

    PubMed

    de Barros Pita, Will; Leite, Fernanda Cristina Bezerra; de Souza Liberal, Anna Theresa; Pereira, Luciana Filgueira; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante; de Morais, Marcos Antonio

    2012-12-01

    The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a 2(-ΔΔC(q)) (comparative C(q) method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.

  7. Characterisation of functional domains in fission yeast Ams2 that are required for core histone gene transcription

    PubMed Central

    Takayama, Yuko; Shirai, Masaki; Masuda, Fumie

    2016-01-01

    Histone gene expression is regulated in a cell cycle-dependent manner, with a peak at S phase, which is crucial for cell division and genome integrity. However, the detailed mechanisms by which expression of histone genes are tightly regulated remain largely unknown. Fission yeast Ams2, a GATA-type zinc finger motif-containing factor, is required for activation of S phase-specific core histone gene transcription. Here we report the molecular characterisation of Ams2. We show that the zinc finger motif in Ams2 is necessary to bind the histone gene promoter region and to activate histone gene transcription. An N-terminal region of Ams2 acts as a self-interaction domain. Intriguingly, N-terminally truncated Ams2 binds to the histone gene promoters, but does not fully activate histone gene transcription. These observations imply that Ams2 self-interactions are required for efficient core histone gene transcription. Moreover, we show that Ams2 interacts with Teb1, which itself binds to the core histone gene promoters. We discuss the relationships between Ams2 domains and efficient transcription of the core histone genes in fission yeast. PMID:27901072

  8. Yeast Microcapsule-Mediated Targeted Delivery of Diverse Nanoparticles for Imaging and Therapy via the Oral Route.

    PubMed

    Zhou, Xing; Zhang, Xiangjun; Han, Songling; Dou, Yin; Liu, Mengyu; Zhang, Lin; Guo, Jiawei; Shi, Qing; Gong, Genghao; Wang, Ruibing; Hu, Jiang; Li, Xiaohui; Zhang, Jianxiang

    2017-02-08

    Targeting of nanoparticles to distant diseased sites after oral delivery remains highly challenging due to the existence of many biological barriers in the gastrointestinal tract. Here we report targeted oral delivery of diverse nanoparticles in multiple disease models, via a "Trojan horse" strategy based on a bioinspired yeast capsule (YC). Diverse charged nanoprobes including quantum dots (QDs), iron oxide nanoparticles (IONPs), and assembled organic fluorescent nanoparticles can be effectively loaded into YC through electrostatic force-driven spontaneous deposition, resulting in different diagnostic YC assemblies. Also, different positive nanotherapies containing an anti-inflammatory drug indomethacin (IND) or an antitumor drug paclitaxel (PTX) are efficiently packaged into YC. YCs containing either nanoprobes or nanotherapies may be rapidly endocytosed by macrophages and maintained in cells for a relatively long period of time. Post oral administration, nanoparticles packaged in YC are first transcytosed by M cells and sequentially endocytosed by macrophages, then transported to neighboring lymphoid tissues, and finally delivered to remote diseased sites of inflammation or tumor in mice or rats, all through the natural route of macrophage activation, recruitment, and deployment. For the examined acute inflammation model, the targeting efficiency of YC-delivered QDs or IONPs is even higher than that of control nanoprobes administered at the same dose via intravenous injection. Assembled IND or PTX nanotherapies orally delivered via YCs exhibit remarkably potentiated efficacies as compared to nanotherapies alone in animal models of inflammation and tumor, which is consistent with the targeting effect and enhanced accumulation of drug molecules at diseased sites. Consequently, through the intricate transportation route, nanoprobes or nanotherapies enveloped in YC can be preferentially delivered to desired targets, affording remarkably improved efficacies for the

  9. The role of the Parkinson's disease gene PARK9 in essential cellular pathways and the manganese homeostasis network in yeast.

    PubMed

    Chesi, Alessandra; Kilaru, Austin; Fang, Xiaodong; Cooper, Antony A; Gitler, Aaron D

    2012-01-01

    YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

  10. Conserved Gene Expression Programs in Developing Roots from Diverse Plants

    PubMed Central

    Huang, Ling; Schiefelbein, John

    2015-01-01

    The molecular basis for the origin and diversification of morphological adaptations is a central issue in evolutionary developmental biology. Here, we defined temporal transcript accumulation in developing roots from seven vascular plants, permitting a genome-wide comparative analysis of the molecular programs used by a single organ across diverse species. The resulting gene expression maps uncover significant similarity in the genes employed in roots and their developmental expression profiles. The detailed analysis of a subset of 133 genes known to be associated with root development in Arabidopsis thaliana indicates that most of these are used in all plant species. Strikingly, this was also true for root development in a lycophyte (Selaginella moellendorffii), which forms morphologically different roots and is thought to have evolved roots independently. Thus, despite vast differences in size and anatomy of roots from diverse plants, the basic molecular mechanisms employed during root formation appear to be conserved. This suggests that roots evolved in the two major vascular plant lineages either by parallel recruitment of largely the same developmental program or by elaboration of an existing root program in the common ancestor of vascular plants. PMID:26265761

  11. Conserved Gene Expression Programs in Developing Roots from Diverse Plants.

    PubMed

    Huang, Ling; Schiefelbein, John

    2015-08-01

    The molecular basis for the origin and diversification of morphological adaptations is a central issue in evolutionary developmental biology. Here, we defined temporal transcript accumulation in developing roots from seven vascular plants, permitting a genome-wide comparative analysis of the molecular programs used by a single organ across diverse species. The resulting gene expression maps uncover significant similarity in the genes employed in roots and their developmental expression profiles. The detailed analysis of a subset of 133 genes known to be associated with root development in Arabidopsis thaliana indicates that most of these are used in all plant species. Strikingly, this was also true for root development in a lycophyte (Selaginella moellendorffii), which forms morphologically different roots and is thought to have evolved roots independently. Thus, despite vast differences in size and anatomy of roots from diverse plants, the basic molecular mechanisms employed during root formation appear to be conserved. This suggests that roots evolved in the two major vascular plant lineages either by parallel recruitment of largely the same developmental program or by elaboration of an existing root program in the common ancestor of vascular plants.

  12. Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes.

    PubMed

    Bianchi, M M; Ngo, S; Vandenbol, M; Sartori, G; Morlupi, A; Ricci, C; Stefani, S; Morlino, G B; Hilger, F; Carignani, G; Slonimski, P P; Frontali, L

    2001-11-01

    Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s).

  13. Introduction of yeast artificial chromosomes containing mutant human amyloid precursor protein genes into transgenic mice

    SciTech Connect

    Call, L.M.; Lamb, B.T.; Boese, K.F.

    1994-09-01

    Several hypothetical mechanisms have been proposed for the generation and deposition of the amyloid beta (A{beta}) peptide in Alzheimer`s disease (AD). These include overexpression of the amyloid precursor protein (APP) gene, as suggested by Down Syndrome (DS, trisomy 21), and mutation of APP, as suggested by mutations associated with the presence of disease/amyloid deposition in some cases of familial AD (FAD). Although numerous in vitro studies have lead to certain insights into the molecular basis for amyloid deposition, the mechanisms(s) of amyloidogenesis in vivo remains poorly defined. To examine the effect of FAD mutations on amyloidogenesis in an animal model, we have focused on producing APP YAC transgenic mice containing the human APP gene with FAD mutations. These APP YAC transgenics are being produced by introduction of a 650 kb APP YAC through lipid-mediated transfection of ES cells. This strategy has two principal advantages: the APP genomic sequences contain transcriptional regulatory elements required for proper spatial and temporal expression and contain appropriate splice donor and acceptor sites needed to generate the entire spectrum of alternatively spliced APP transcripts. As a first step, we cloned the genomic regions surrounding APP exons 16 and 17 from an APP YAC sublibrary. Both the Swedish and the 717 mutations were then introduced into exons 16 and 17, respectively, by PCR mutagenesis, and subsequently transferred into the 650 kb APP YAC by a two step gene replacement in yeast. The mutant YACs have been introduced into ES cells, and we have determined that these cells are expressing human mutant APP mRNA and protein. These cells are being used to generate transgenic mice. This paradigm should provide the appropriate test of whether a mutant APP gene is capable of producing AD-like pathology in a mouse model.

  14. Increased biomass production of industrial bakers' yeasts by overexpression of Hap4 gene.

    PubMed

    Dueñas-Sánchez, Rafael; Codón, Antonio C; Rincón, Ana M; Benítez, Tahía

    2010-10-15

    HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomyces cerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' industry. Invertase (Suc2p) and maltase (Mal12p) activities, growth and sugar consumption rates in laboratory (YPD) or industrial media (MAB) were also comparable in bakers' strains and their transformants, but VTH, DTH and 3TH increased their final biomass production by 9.5, 5.0 and 5.0% respectively as compared to their parentals in MAB. Furthermore, V1 and its transformant VTH had comparable capacity to ferment lean doughs (volume increase rate and final volume) while V3 and its transformant 3TH fermented sweet doughs in a similar manner. Therefore transformants possessed increased biomass yield and appropriate characteristics to be employed in bakers' industry because they lacked drug resistant markers and bacterial DNA, and were genetically stable. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene.

    PubMed

    Ali, Shawkat; Mannan, Abdul; El Oirdi, Mohamed; Waheed, Abdul; Mirza, Bushra

    2012-06-12

    Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L(-1) along with 250 mg L(-1) each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L(-1) of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.

  16. Identification of New Genes Involved in the Regulation of Yeast Alcohol Dehydrogenase II

    PubMed Central

    Denis, Clyde L.

    1984-01-01

    Recessive mutations in two negative control elements, CRE1 and CRE2, have been obtained that allow the glucose-repressible alcohol dehydrogenase (ADHII) of yeast to escape repression by glucose. Both the cre1 and cre2 alleles affected ADHII synthesis irrespective of the allele of the positive effector, ADR1. However, for complete derepression of ADHII synthesis, a wild-type ADR1 gene was required. Neither the cre1 nor cre2 alleles affected the expression of several other glucose-repressible enzymes. A third locus, CCR4, was identified by recessive mutations that suppressed the cre1 and cre2 phenotypes. The ccr4 allele blocked the derepression of ADHII and several other glucose-repressible enzymes, indicating that the CCR4 gene is a positive control element. The ccr4 allele had no effect on the repression of ADHII when it was combined with the ADR1-5c allele, whereas the phenotypically similar ccr1 allele, which partially suppresses ADR1-5c, did not suppress the cre1 or cre2 phenotype. Complementation studies also indicated that ccr1 and snf1 are allelic. A model of ADHII regulation is proposed in which both ADR1 and CCR4 are required for ADHII expression. CRE1 and CRE2 negatively control CCR4, whereas CCR1 is required for ADR1 function. PMID:6392016

  17. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene

    PubMed Central

    2012-01-01

    Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1 along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated. PMID:22691292

  18. Nuclear RNA Decay Pathways Aid Rapid Remodeling of Gene Expression in Yeast.

    PubMed

    Bresson, Stefan; Tuck, Alex; Staneva, Desislava; Tollervey, David

    2017-03-02

    In budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprogramming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV crosslinking immediately following glucose withdrawal (0, 4, and 8 min). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many growth-related mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. We conclude that transcription termination is followed by TRAMP-mediated RNA decay. Upregulated transcripts evaded increased surveillance factor binding following glucose withdrawal. Some upregulated genes showed use of alternative transcription starts to bypass strong NNS binding sites. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability.

  19. Genes required for initiation and resolution steps of mating-type switching in fission yeast.

    PubMed

    Egel, R; Beach, D H; Klar, A J

    1984-06-01

    The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA derived from either of the two storage cassettes, mat2 -P and mat3 -M, into the expression locus, mat1 . The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat1 . Fifty-three mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat1 , which reduce the amount of the DNA double-strand cut. The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three ( swi1 , -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes ( swi4 , -8, -9) undergo high-frequency rearrangement of the mating-type locus indicative of errors of resolution of recombinational intermediates. The remaining three ( swi2 , -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps.

  20. Gene activation by copy transposition in mating-type switching of a homothallic fission yeast.

    PubMed

    Egel, R; Gutz, H

    1981-04-01

    Mating-type switching in homothallic clones of the fission yeast, Schizosaccharomyces pombe, appears to follow the same route as previously found for "mutations" from homothallism to heterothallic ⊕ strains. A copy of mat2-P is transposed to and inserted at mat1, where it functionally replaces the mat1-M allele, and only the mat1 segment is expressed (!) to determine the actual mating type: mat1-M(!) mat2-P = ⊖ ⇌ ⊕ = mat1-P(!) mat2-P. This phenomenon has hitherto been concealed by the high switch-back rate from ⊕ to ⊖ observed in homothallic wild-type strains. It only becomes apparent in the presence of mutant "switching genes", which retard the rates of mating-type interconversion and temporarily freeze one or the other state of gene activation at the mat1 segment. Mutations to lowered rates of switching are found to map both inside and outside the mating-type locus. While the internal mutations of this kind exert their effect autonomously in the cis-configuration, the unlinked mutations are recessive to their wild-type alleles.

  1. Genes required for initiation and resolution steps of mating-type switching in fission yeast.

    PubMed Central

    Egel, R; Beach, D H; Klar, A J

    1984-01-01

    The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA derived from either of the two storage cassettes, mat2 -P and mat3 -M, into the expression locus, mat1 . The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat1 . Fifty-three mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat1 , which reduce the amount of the DNA double-strand cut. The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three ( swi1 , -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes ( swi4 , -8, -9) undergo high-frequency rearrangement of the mating-type locus indicative of errors of resolution of recombinational intermediates. The remaining three ( swi2 , -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps. Images PMID:6587363

  2. Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

    PubMed Central

    Hegde, Vijay; Klein, Hannah

    2000-01-01

    Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism. PMID:10908335

  3. Origin and evolution of laminin gene family diversity.

    PubMed

    Fahey, Bryony; Degnan, Bernard M

    2012-07-01

    Laminins are a family of multidomain glycoproteins that are important contributors to the structure of metazoan extracellular matrices. To investigate the origin and evolution of the laminin family, we characterized the full complement of laminin-related genes in the genome of the sponge, Amphimedon queenslandica. As a representative of the Demospongiae, a group consistently placed within the earliest diverging branch of animals by molecular phylogenies, Amphimedon is uniquely placed to provide insight into early steps in the evolution of metazoan gene families. Five Amphimedon laminin-related genes possess the conserved molecular features, and most of the domains found in bilaterian laminins, but all display domain architectures distinct from those of the canonical laminin chain types known from model bilaterians. This finding prompted us to perform a comparative genomic analysis of laminins and related genes from a choanoflagellate and diverse metazoans and to conduct phylogenetic analyses using the conserved Laminin N-terminal domain in order to explore the relationships between genes with distinct architectures. Laminin-like genes appear to have originated in the holozoan lineage (choanoflagellates + metazoans + several other unicellular opisthokont taxa), with several laminin domains originating later and appearing only in metazoan (animal) or eumetazoan (placozoans + ctenophores + cnidarians + bilaterians) laminins. Typical bilaterian α, β, and γ laminin chain forms arose in the eumetazoan stem and another chain type that is conserved in Amphimedon, the cnidarian, Nematostella vectensis, and the echinoderm, Strongylocentrotus purpuratus, appears to have been lost independently from the placozoan, Trichoplax adhaerens, and from multiple bilaterians. Phylogenetic analysis did not clearly reconstruct relationships between the distinct laminin chain types (with the exception of the α chains) but did reveal how several members of the netrin family were

  4. Diversity, evolution, and horizontal gene transfer (HGT) in soda lakes

    NASA Astrophysics Data System (ADS)

    Pinkart, Holly C.; Storrie-Lombardi, Michael C.

    2007-09-01

    Soap Lake is a hypersaline, alkaline lake in Central Washington State (USA). For the past five years the lake has been the site of an NSF Microbial Observatory project devoted to identifying critical geochemical and microbial characteristics of the monimolimnion sediment and water column, and has demonstrated rich multispecies communities occupy all areas of the lake. Soap Lake and similar soda lakes are subject to repeated transient periods of extreme evaporation characterized by significant repetitive alterations in salinity, pH, and total water volume, yet maintain high genetic and metabolic diversity. It has been argued that this repetitive cycle for salinity, alkalinity, and sulfur concentration has been a major driver for prokaryote evolution and diversity. The rapidity of wet-dry cycling places special demands on genome evolution, requirements that are beyond the relatively conservative eukaryotic evolutionary strategy of serial alteration of existing gene sequences in a relatively stable genome. Although HGT is most likely responsible for adding a significant amount of noise to the genetic record, analysis of HGT activity can also provide us with a much-needed probe for exploration of prokaryotic genome evolution and the origin of diversity. Packaging of genetic information within the protective protein capsid of a bacteriophage would seem preferable to exposing naked DNA to the highly alkaline conditions in the lake. In this study, we present preliminary data demonstrating the presence of a d