Sample records for yeast 2-hybrid screen

  1. Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

    PubMed Central

    Kalay, Gizem; Lusk, Richard; Dome, Mackenzie; Hens, Korneel; Deplancke, Bart; Wittkopp, Patricia J.

    2016-01-01

    The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. PMID:27527791

  2. [Identification of C(2)M interacting proteins by yeast two-hybrid screening].

    PubMed

    Yue, Shan-shan; Xia, Lai-xin

    2015-11-01

    The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

  3. Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

    PubMed

    Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

    2018-02-16

    Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

  4. The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration.

    PubMed

    Abuqarn, Mehtap; Allmeling, Christina; Amshoff, Inga; Menger, Bjoern; Nasser, Inas; Vogt, Peter M; Reimers, Kerstin

    2011-07-01

    Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration. 2011 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-09-01

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

  6. Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

    PubMed

    Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

    2018-01-16

    "A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

  7. A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

    PubMed Central

    Swick, Lance; Kapatos, Gregory

    2008-01-01

    The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5′-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal α-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal α-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal α–helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1–96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1–42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1–42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1–GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4. PMID:16696853

  8. Screening of binding proteins that interact with Chinese sacbrood virus VP3 capsid protein in Apis cerana larvae cDNA library by the yeast two-hybrid method.

    PubMed

    Fei, Dongliang; Wei, Dong; Yu, Xiaolei; Yue, Jinjin; Li, Ming; Sun, Li; Jiang, Lili; Li, Yijing; Diao, Qingyun; Ma, Mingxiao

    2018-03-15

    Chinese sacbrood virus (CSBV) causes larval death and apiary collapse of Apis cerana. VP3 is a capsid protein of CSBV but its function is poorly understood. To determine the function of VP3 and screen for novel binding proteins that interact with VP3, we conducted yeast two-hybrid screening, glutathione S-transferase pull-down, and co-immunoprecipitation assays. Galectin (GAL) is a protein involved in immune regulation and host-pathogen interactions. The yeast two-hybrid screen implicated GAL as a major VP3-binding candidate. The assays showed that the VP3 interacted with GAL. Identification of these cellular targets and clarifying their contributions to the host-pathogen interaction may be useful for the development of novel therapeutic and prevention strategies against CSBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Mapping protein-protein interactions using yeast two-hybrid assays.

    PubMed

    Mehla, Jitender; Caufield, J Harry; Uetz, Peter

    2015-05-01

    Yeast two-hybrid (Y2H) screens are an efficient system for mapping protein-protein interactions and whole interactomes. The screens can be performed using random libraries or collections of defined open reading frames (ORFs) called ORFeomes. This protocol describes both library and array-based Y2H screening, with an emphasis on array-based assays. Array-based Y2H is commonly used to test a number of "prey" proteins for interactions with a single "bait" (target) protein or pool of proteins. The advantage of this approach is the direct identification of interacting protein pairs without further downstream experiments: The identity of the preys is known and does not require further confirmation. In contrast, constructing and screening a random prey library requires identification of individual prey clones and systematic retesting. Retesting is typically performed in an array format. © 2015 Cold Spring Harbor Laboratory Press.

  10. Novel brewing yeast hybrids: creation and application.

    PubMed

    Krogerus, Kristoffer; Magalhães, Frederico; Vidgren, Virve; Gibson, Brian

    2017-01-01

    The natural interspecies Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid yeast is responsible for global lager beer production and is one of the most important industrial microorganisms. Its success in the lager brewing environment is due to a combination of traits not commonly found in pure yeast species, principally low-temperature tolerance, and maltotriose utilization. Parental transgression is typical of hybrid organisms and has been exploited previously for, e.g., the production of wine yeast with beneficial properties. The parental strain S. eubayanus has only been discovered recently and newly created lager yeast strains have not yet been applied industrially. A number of reports attest to the feasibility of this approach and artificially created hybrids are likely to have a significant impact on the future of lager brewing. De novo S. cerevisiae × S. eubayanus hybrids outperform their parent strains in a number of respects, including, but not restricted to, fermentation rate, sugar utilization, stress tolerance, and aroma formation. Hybrid genome function and stability, as well as different techniques for generating hybrids and their relative merits are discussed. Hybridization not only offers the possibility of generating novel non-GM brewing yeast strains with unique properties, but is expected to aid in unraveling the complex evolutionary history of industrial lager yeast.

  11. New lager yeast strains generated by interspecific hybridization.

    PubMed

    Krogerus, Kristoffer; Magalhães, Frederico; Vidgren, Virve; Gibson, Brian

    2015-05-01

    The interspecific hybrid Saccharomyces pastorianus is the most commonly used yeast in brewery fermentations worldwide. Here, we generated de novo lager yeast hybrids by mating a domesticated and strongly flocculent Saccharomyces cerevisiae ale strain with the Saccharomyces eubayanus type strain. The hybrids were characterized with respect to the parent strains in a wort fermentation performed at temperatures typical for lager brewing (12 °C). The resulting beers were analysed for sugar and aroma compounds, while the yeasts were tested for their flocculation ability and α-glucoside transport capability. These hybrids inherited beneficial properties from both parent strains (cryotolerance, maltotriose utilization and strong flocculation) and showed apparent hybrid vigour, fermenting faster and producing beer with higher alcohol content (5.6 vs 4.5 % ABV) than the parents. Results suggest that interspecific hybridization is suitable for production of novel non-GM lager yeast strains with unique properties and will help in elucidating the evolutionary history of industrial lager yeast.

  12. Precision and recall estimates for two-hybrid screens

    PubMed Central

    Huang, Hailiang; Bader, Joel S.

    2009-01-01

    Motivation: Yeast two-hybrid screens are an important method to map pairwise protein interactions. This method can generate spurious interactions (false discoveries), and true interactions can be missed (false negatives). Previously, we reported a capture–recapture estimator for bait-specific precision and recall. Here, we present an improved method that better accounts for heterogeneity in bait-specific error rates. Result: For yeast, worm and fly screens, we estimate the overall false discovery rates (FDRs) to be 9.9%, 13.2% and 17.0% and the false negative rates (FNRs) to be 51%, 42% and 28%. Bait-specific FDRs and the estimated protein degrees are then used to identify protein categories that yield more (or fewer) false positive interactions and more (or fewer) interaction partners. While membrane proteins have been suggested to have elevated FDRs, the current analysis suggests that intrinsic membrane proteins may actually have reduced FDRs. Hydrophobicity is positively correlated with decreased error rates and fewer interaction partners. These methods will be useful for future two-hybrid screens, which could use ultra-high-throughput sequencing for deeper sampling of interacting bait–prey pairs. Availability: All software (C source) and datasets are available as supplemental files and at http://www.baderzone.org under the Lesser GPL v. 3 license. Contact: joel.bader@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19091773

  13. Yeast-2-Hybrid data file showing progranulin interactions in human fetal brain and bone marrow libraries.

    PubMed

    Tegeder, Irmgard

    2016-12-01

    Progranulin deficiency in humans is associated with neurodegeneration. Its mechanisms are not yet fully understood. We performed a Yeast-2-Hybrid screen using human full-length progranulin as bait to assess the interactions of progranulin. Progranulin was screened against human fetal brain and human bone marrow libraries using the standard Matchmaker technology (Clontech). This article contains the full Y2H data table, including blast results and sequences, a sorted table according to selection criteria for likely positive, putatively positive, likely false and false preys, and tables showing the gene ontology terms associated with the likely and putative preys of the brain and bone marrow libraries. The interactions with autophagy proteins were confirmed and functionally analyzed in "Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy" (C. Altmann, S. Hardt, C. Fischer, J. Heidler, H.Y. Lim, A. Haussler, B. Albuquerque, B. Zimmer, C. Moser, C. Behrends, F. Koentgen, I. Wittig, M.H. Schmidt, A.M. Clement, T. Deller, I. Tegeder, 2016) [1].

  14. Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains.

    PubMed

    El-Fiky, Zaki A; Hassan, Gamal M; Emam, Ahmed M

    2012-06-01

    Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories. © 2012 Institute of Food Technologists®

  15. T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium, mercury, and zinc.

    PubMed

    Li, Jian; Liu, Yun; Kong, Dongdong; Ren, Shujuan; Li, Na

    2016-05-01

    In the present study, a two-hybrid yeast bioassay and a T-screen were used to screen for the thyroid receptor (TR)-disrupting activity of select metallic compounds (CdCl2, ZnCl2, HgCl2, CuSO4, MnSO4, and MgSO4). The results reveal that none of the tested metallic compounds showed TR-agonistic activity, whereas ZnCl2, HgCl2, and CdCl2 demonstrated TR antagonism. For the yeast assay, the dose-response relationship of these metallic compounds was established, and the concentrations producing 20 % of the maximum effect of ZnCl2, HgCl2, and CdCl2 were 9.1 × 10(-5), 3.2 × 10(-6), and 1.2 × 10(-6) mol/L, respectively. The T-screen also supported the finding that ZnCl2, HgCl2, and CdCl2 decreased the cell proliferation at concentrations ranging from 10(-6) to 10(-4) mol/L. Furthermore, the thyroid-disrupting activity of metallic compounds in environmental water samples collected from the Guanting Reservoir, Beijing, China was evaluated. Solid-phase extraction was used to separate the organic extracts, and a modified two-hybrid yeast bioassay revealed that the metallic compounds in the water samples could affect thyroid hormone-induced signaling by decreasing the binding of the thyroid hormone. The addition of ethylenediaminetetraacetic acid (30 mg/L) could eliminate the effects. Thus, the cause(s) of the thyroid toxicity in the water samples appeared to be partly related to the metallic compounds.

  16. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

  17. A strategy for rapid production and screening of yeast artificial chromosome libraries.

    PubMed

    Strauss, W M; Jaenisch, E; Jaenisch, R

    1992-01-01

    We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

  18. Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.

    PubMed

    Odell, Anahi V; Tran, Fanny; Foderaro, Jenna E; Poupart, Séverine; Pathak, Ravi; Westwood, Nicholas J; Ward, Gary E

    2015-01-01

    Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

  19. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

    PubMed

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

    2012-01-01

    Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

  20. Identification of proteins interacting with Toxoplasma SRCAP by yeast two-hybrid screening.

    PubMed

    Nallani, Karuna C; Sullivan, William J

    2005-03-01

    Toxoplasma gondii is an opportunistic protozoan parasite that differentiates into latent cysts (bradyzoite) that can be reactivated during immunosuppression. TgSRCAP (Toxoplasma gondii Snf2-related CBP activator protein) is a SWI2/SNF2 family chromatin remodeler whose expression increases during cyst development. Identifying the proteins associating with TgSRCAP during the pre-cyst stage (tachyzoite) will increase our understanding of how parasite differentiation is initiated. We employed the yeast two-hybrid system to identify proteins that may interact directly with TgSRCAP. A stretch of 1,060 amino acids between ATPase subdomains IV and V of TgSRCAP was chosen as "bait" since the corresponding region in human SRCAP interacts with other proteins, including CREB binding protein. We have identified several novel parasite-specific transcription factors predicted to be in the T. gondii genome. Metabolic enzymes that may participate in cyst development were also identified as interacting with TgSRCAP.

  1. Yeast one-hybrid screening the potential regulator of CYP6B6 overexpression of Helicoverpa armigera under 2-tridecanone stress.

    PubMed

    Zhao, J; Liu, X N; Li, F; Zhuang, S Z; Huang, L N; Ma, J; Gao, X W

    2016-04-01

    In insect, the cytochrome P450 plays a pivotal role in detoxification to toxic allelochemicals. Helicoverpa armigera can tolerate and survive in 2-tridecanone treatment owing to the CYP6B6 responsive expression, which is controlled by some regulatory DNA sequences and transcription regulators. Therefore, the 2-tridecanone responsive region and transcription regulators of the CYP6B6 are responsible for detoxification of cotton bollworm. In this study, we used yeast one-hybrid to screen two potential transcription regulators of the CYP6B6 from H. armigera that respond to the plant secondary toxicant 2-tridecanone, which were named Prey1 and Prey2, respectively. According to the NCBI database blast, Prey1 is the homology with FK506 binding protein (FKBP) of Manduca sexta and Bombyx mori that belongs to the FKBP-C superfamily, while Prey2 may be a homology of an unknown protein of Papilio or the fcaL24 protein homology of B. mori. The electrophoretic mobility shift assays revealed that the FKBP of prokaryotic expression could specifically bind to the active region of the CYP6B6 promoter. After the 6th instar larvae of H. armigera reared on 2-tridecanone artificial diet, we found there were similar patterns of CYP6B6 and FKBP expression of the cotton bollworm treated with 10 mg g-1 2-tridecanone for 48 h, which correlation coefficient was the highest (0.923). Thus, the FKBP is identified as a strong candidate for regulation of the CYP6B6 expression, when the cotton bollworm is treated with 2-tridecanone. This may lead us to a better understanding of transcriptional mechanism of CYP6B6 and provide very useful information for the pest control.

  2. Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

    PubMed Central

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

    2012-01-01

    Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

  3. Identification of novel transcriptional regulators of Zat12 using comprehensive yeast one-hybrid screens.

    PubMed

    Ben Daniel, Bat-Hen; Cattan, Esther; Wachtel, Chaim; Avrahami, Dorit; Glick, Yair; Malichy, Asaf; Gerber, Doron; Miller, Gad

    2016-08-01

    To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses. © 2016 Scandinavian Plant Physiology Society.

  4. Evolutionary Role of Interspecies Hybridization and Genetic Exchanges in Yeasts

    PubMed Central

    Dujon, Bernard

    2012-01-01

    Summary: Forced interspecific hybridization has been used in yeasts for many years to study speciation or to construct artificial strains with novel fermentative and metabolic properties. Recent genome analyses indicate that natural hybrids are also generated spontaneously between yeasts belonging to distinct species, creating lineages with novel phenotypes, varied genetic stability, or altered virulence in the case of pathogens. Large segmental introgressions from evolutionarily distant species are also visible in some yeast genomes, suggesting that interspecific genetic exchanges occur during evolution. The origin of this phenomenon remains unclear, but it is likely based on weak prezygotic barriers, limited Dobzhansky-Muller (DM) incompatibilities, and rapid clonal expansions. Newly formed interspecies hybrids suffer rapid changes in the genetic contribution of each parent, including chromosome loss or aneuploidy, translocations, and loss of heterozygosity, that, except in a few recently studied cases, remain to be characterized more precisely at the genomic level by use of modern technologies. We review here known cases of natural or artificially formed interspecies hybrids between yeasts and discuss their potential importance in terms of genome evolution. Problems of meiotic fertility, ploidy constraint, gene and gene product compatibility, and nucleomitochondrial interactions are discussed and placed in the context of other known mechanisms of yeast genome evolution as a model for eukaryotes. PMID:23204364

  5. Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

    PubMed

    Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

    2014-01-01

    Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

  6. Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen.

    PubMed

    Santiago, Ednalise; Akamine, Pearl; Snider, Jamie; Wong, Victoria; Jessulat, Matthew; Deineko, Viktor; Gagarinova, Alla; Aoki, Hiroyuki; Minic, Zoran; Phanse, Sadhna; San Antonio, Andrea; Cubano, Luis A; Rymond, Brian C; Babu, Mohan; Stagljar, Igor; Rodriguez-Medina, Jose R

    2016-05-03

    Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis. Copyright © 2016 Santiago et al.

  7. Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system.

    PubMed

    Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

    2006-01-01

    To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease.

  8. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

    PubMed Central

    2011-01-01

    Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

  9. Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

    PubMed

    Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

    2011-10-20

    The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

  10. A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

    PubMed

    Lev, Ifat; Shemesh, Keren; Volpe, Marina; Sau, Soumitra; Levinton, Nelly; Molco, Maya; Singh, Shivani; Liefshitz, Batia; Ben Aroya, Shay; Kupiec, Martin

    2017-07-01

    The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans -acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA. Copyright © 2017 by the Genetics Society of America.

  11. Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system

    PubMed Central

    Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Yamada, Hisashi; Horiguchi-Yamada, Junko

    2006-01-01

    OBJECTIVE To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection. METHODS Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture. RESULTS Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion. CONCLUSION The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease. PMID:18651010

  12. Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

    PubMed

    Reuter, T; Herterich, S; Bernhard, O; Hoehn, H; Gross, H J

    2000-01-15

    Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

  13. Evaluation of a recombinant yeast cell estrogen screening assay.

    PubMed Central

    Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

    1997-01-01

    A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720

  14. 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.

  15. Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

    PubMed Central

    Gladue, Douglas P.; Baker-Bransetter, Ryan; Holinka, Lauren G.; Fernandez-Sainz, Ignacio J.; O’Donnell, Vivian; Fletcher, Paige; Lu, Zhiqiang; Borca, Manuel V.

    2014-01-01

    E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle. PMID:24416391

  16. Yeast One-Hybrid Gγ Recruitment System for Identification of Protein Lipidation Motifs

    PubMed Central

    Fukuda, Nobuo; Doi, Motomichi; Honda, Shinya

    2013-01-01

    Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs. PMID:23922919

  17. Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae.

    PubMed

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

    2013-01-01

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

  18. Saccharomyces interspecies hybrids as model organisms for studying yeast adaptation to stressful environments.

    PubMed

    Lopandic, Ksenija

    2018-01-01

    The strong development of molecular biology techniques and next-generation sequencing technologies in the last two decades has significantly improved our understanding of the evolutionary history of Saccharomyces yeasts. It has been shown that many strains isolated from man-made environments are not pure genetic lines, but contain genetic materials from different species that substantially increase their genome complexity. A number of strains have been described as interspecies hybrids, implying different yeast species that under specific circumstances exchange and recombine their genomes. Such fusing usually results in a wide variety of alterations at the genetic and chromosomal levels. The observed changes have suggested a high genome plasticity and a significant role of interspecies hybridization in the adaptation of yeasts to environmental stresses and industrial processes. There is a high probability that harsh wine and beer fermentation environments, from which the majority of interspecies hybrids have been isolated so far, influence their selection and stabilization as well as their genomic and phenotypic heterogeneity. The lessons we have learned about geno- and phenotype plasticity and the diversity of natural and commercial yeast hybrids have already had a strong impact on the development of artificial hybrids that can be successfully used in the fermentation-based food and beverage industry. The creation of artificial hybrids through the crossing of strains with desired attributes is a possibility to obtain a vast variety of new, but not genetically modified yeasts with a range of improved and beneficial traits. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

    PubMed

    Leducq, Jean-Baptiste; Nielly-Thibault, Lou; Charron, Guillaume; Eberlein, Chris; Verta, Jukka-Pekka; Samani, Pedram; Sylvester, Kayla; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

    2016-01-11

    Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

  20. Genome sequence of the lager brewing yeast, an interspecies hybrid.

    PubMed

    Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

    2009-04-01

    This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

  1. Genome Sequence of the Lager Brewing Yeast, an Interspecies Hybrid

    PubMed Central

    Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

    2009-01-01

    This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production. PMID:19261625

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

    PubMed Central

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

    2013-01-01

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

  3. Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction.

    PubMed

    Zhou, Jingwen; Zhou, Haiyan; Du, Guocheng; Liu, Liming; Chen, Jian

    2010-09-01

    To obtain a thiamine-auxotrophic yeast strain that overproduces alpha-ketoglutaric acid (alpha-KG) from glycerol and to investigate nutrient effects on alpha-KG production. Yeast strain WSH-Z06, a thiamine auxotroph that gave high yields of alpha-KG from glycerol, was obtained by screening for ampicillin/kanamycin resistance and thiamine auxotrophy. The strain was identified as Yarrowia lipolytica based on physiological, chemical, and phylogenetic analysis. The ability of the strain to convert glycerol to alpha-KG was analysed by investigating the effects of nutritional factors, including thiamine, riboflavin, nitrogen sources, and calcium ion. Thiamine and calcium ion concentration had the greatest effect on alpha-KG accumulation. Under optimal conditions, a yield of 39.2 g l(-1)alpha-KG was obtained from 100 g l(-1) glycerol, with 16.84 g l(-1) pyruvate as a by-product. The current work provides a method for screening for an alpha-KG overproducer. Nutrients have a significant impact on alpha-KG production in the yeast strain presented here. The alpha-KG-overproducing yeast strain Y. lipolytica WSH-Z06 is a promising parent strain for further metabolic engineering to lower by-product accumulation and accelerate glycerol utilization.

  4. Network topological analysis reveals the functional cohesiveness for the newly discovered links by Yeast 2 Hybrid approach

    NASA Astrophysics Data System (ADS)

    Ghiassian, Susan; Pevzner, Sam; Rolland, Thomas; Tassan, Murat; Barabasi, Albert Laszlo; Vidal, Mark; CCNR, Northeastern University Collaboration; Dana Farber Cancer Institute Collaboration

    2014-03-01

    Protein-protein interaction maps and interactomes are the blueprint of Network Medicine and systems biology and are being experimentally studied by different groups. Despite the wide usage of Literature Curated Interactome (LCI), these sources are biased towards different parameters such as highly studied proteins. Yeast two hybrid method is a high throughput experimental setup which screens proteins in an unbiased fashion. Current knowledge of protein interactions is far from complete. In fact the previous offered data from Y2H method (2005), is estimated to offer only 5% of all potential protein interactions. Currently this coverage has increased to 20% of what is known as reference HI In this work we study the topological properties of Y2H protein-protein interactions network with LCI and show although they both agree on some properties, LCI shows a clear unbiased nature of interaction selections. Most importantly, we assess the properties of PPI as it evolves with increasing the coverage. We show that, the newly discovered interactions tend to connect proteins that have been closer than average in the previous PPI release. reinforcing the modular structure of PPI. Furthermore, we show, some unseen effects on PPI (as opposed to LCI) can be explained by its incompleteness.

  5. Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system.

    PubMed

    Hussey, Stephen L; Muddana, Smita S; Peterson, Blake R

    2003-04-02

    Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

  6. A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology.

    PubMed

    Yu, Deshui; Liao, Libing; Zhang, Ju; Zhang, Yi; Xu, Kedong; Liu, Kun; Li, Xiaoli; Tan, Guangxuan; Chen, Ran; Wang, Yulu; Liu, Xia; Zhang, Xuan; Han, Xiaomeng; Wei, Zhangkun; Li, Chengwei

    2018-05-01

    Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

  7. Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii

    PubMed Central

    2017-01-01

    ABSTRACT Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae. Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression

  8. Use of The Yeast Two-Hybrid System to Identify Targets of Fungal Effectors

    USDA-ARS?s Scientific Manuscript database

    The yeast-two hybrid (Y2H) system is a binary method widely used to determine direct interactions between paired proteins. Although having certain limitations, this method has become one of the two main systemic tools (along with affinity purification/mass spectrometry) for interactome mapping in mo...

  9. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

    PubMed

    Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

    1994-12-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

  10. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast

    PubMed Central

    1994-01-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine- nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  11. Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii.

    PubMed

    Ortiz-Merino, Raúl A; Kuanyshev, Nurzhan; Byrne, Kevin P; Varela, Javier A; Morrissey, John P; Porro, Danilo; Wolfe, Kenneth H; Branduardi, Paola

    2018-03-01

    Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns

  12. Characterization of single chain antibody targets through yeast two hybrid

    PubMed Central

    2010-01-01

    Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear

  13. Combined zebrafish-yeast chemical-genetic screens reveal gene-copper-nutrition interactions that modulate melanocyte pigmentation.

    PubMed

    Ishizaki, Hironori; Spitzer, Michaela; Wildenhain, Jan; Anastasaki, Corina; Zeng, Zhiqiang; Dolma, Sonam; Shaw, Michael; Madsen, Erik; Gitlin, Jonathan; Marais, Richard; Tyers, Mike; Patton, E Elizabeth

    2010-01-01

    Hypopigmentation is a feature of copper deficiency in humans, as caused by mutation of the copper (Cu(2+)) transporter ATP7A in Menkes disease, or an inability to absorb copper after gastric surgery. However, many causes of copper deficiency are unknown, and genetic polymorphisms might underlie sensitivity to suboptimal environmental copper conditions. Here, we combined phenotypic screens in zebrafish for compounds that affect copper metabolism with yeast chemical-genetic profiles to identify pathways that are sensitive to copper depletion. Yeast chemical-genetic interactions revealed that defects in intracellular trafficking pathways cause sensitivity to low-copper conditions; partial knockdown of the analogous Ap3s1 and Ap1s1 trafficking components in zebrafish sensitized developing melanocytes to hypopigmentation in low-copper environmental conditions. Because trafficking pathways are essential for copper loading into cuproproteins, our results suggest that hypomorphic alleles of trafficking components might underlie sensitivity to reduced-copper nutrient conditions. In addition, we used zebrafish-yeast screening to identify a novel target pathway in copper metabolism for the small-molecule MEK kinase inhibitor U0126. The zebrafish-yeast screening method combines the power of zebrafish as a disease model with facile genome-scale identification of chemical-genetic interactions in yeast to enable the discovery and dissection of complex multigenic interactions in disease-gene networks.

  14. Loss of Heterozygosity Drives Adaptation in Hybrid Yeast.

    PubMed

    Smukowski Heil, Caiti S; DeSevo, Christopher G; Pai, Dave A; Tucker, Cheryl M; Hoang, Margaret L; Dunham, Maitreya J

    2017-07-01

    Hybridization is often considered maladaptive, but sometimes hybrids can invade new ecological niches and adapt to novel or stressful environments better than their parents. The genomic changes that occur following hybridization that facilitate genome resolution and/or adaptation are not well understood. Here, we examine hybrid genome evolution using experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae × Saccharomyces uvarum and their parentals. We evolved these strains in nutrient-limited conditions for hundreds of generations and sequenced the resulting cultures identifying numerous point mutations, copy number changes, and loss of heterozygosity (LOH) events, including species-biased amplification of nutrient transporters. We focused on a particularly interesting example, in which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecific hybrids. Using allele replacement methods, we tested the fitness of different alleles in hybrid and S. cerevisiae strain backgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S. cerevisiae and the hybrids. This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosity and demonstrates that even infrequent outcrossing may have lasting impacts on adaptation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Nutrient supplements boost yeast transformation efficiency

    PubMed Central

    Yu, Sheng-Chun; Dawson, Alexander; Henderson, Alyssa C.; Lockyer, Eloise J.; Read, Emily; Sritharan, Gayathri; Ryan, Marjah; Sgroi, Mara; Ngou, Pok M.; Woodruff, Rosie; Zhang, Ruifeng; Ren Teen Chia, Travis; Liu, Yu; Xiang, Yiyu; Spanu, Pietro D.

    2016-01-01

    Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per μg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening. PMID:27760994

  16. Yeast Two-Hybrid: State of the Art

    PubMed Central

    Beyaert, Rudi

    1999-01-01

    Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination. PMID:12734586

  17. Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

    PubMed

    Nilles, Matthew L

    2017-01-01

    Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

  18. Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

    PubMed

    Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

    2018-01-01

    Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

    PubMed Central

    Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

    2010-01-01

    Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

  20. Engineered yeast for enhanced CO2 mineralization†

    PubMed Central

    Barbero, Roberto; Carnelli, Lino; Simon, Anna; Kao, Albert; Monforte, Alessandra d’Arminio; Riccò, Moreno; Bianchi, Daniele; Belcher, Angela

    2014-01-01

    In this work, a biologically catalyzed CO2 mineralization process for the capture of CO2 from point sources was designed, constructed at a laboratory scale, and, using standard chemical process scale-up protocols, was modeled and evaluated at an industrial scale. A yeast display system in Saccharomyces cerevisae was used to screen several carbonic anhydrase isoforms and mineralization peptides for their impact on CO2 hydration, CaCO3 mineralization, and particle settling rate. Enhanced rates for each of these steps in the CaCO3 mineralization process were confirmed using quantitative techniques in lab-scale measurements. The effect of these enhanced rates on the CO2 capture cost in an industrial scale CO2 mineralization process using coal fly ash as the CaO source was evaluated. The model predicts a process using bCA2- yeast and fly ash is ~10% more cost effective per ton of CO2 captured than a process with no biological molecules, a savings not realized by wild-type yeast and high-temperature stable recombinant CA2 alone or in combination. The levelized cost of electricity for a power plant using this process was calculated and scenarios in which this process compares favorably to CO2 capture by MEA absorption process are presented. PMID:25289021

  1. Database for High Throughput Screening Hits (dHITS): a simple tool to retrieve gene specific phenotypes from systematic screens done in yeast.

    PubMed

    Chuartzman, Silvia G; Schuldiner, Maya

    2018-03-25

    In the last decade several collections of Saccharomyces cerevisiae yeast strains have been created. In these collections every gene is modified in a similar manner such as by a deletion or the addition of a protein tag. Such libraries have enabled a diversity of systematic screens, giving rise to large amounts of information regarding gene functions. However, often papers describing such screens focus on a single gene or a small set of genes and all other loci affecting the phenotype of choice ('hits') are only mentioned in tables that are provided as supplementary material and are often hard to retrieve or search. To help unify and make such data accessible, we have created a Database of High Throughput Screening Hits (dHITS). The dHITS database enables information to be obtained about screens in which genes of interest were found as well as the other genes that came up in that screen - all in a readily accessible and downloadable format. The ability to query large lists of genes at the same time provides a platform to easily analyse hits obtained from transcriptional analyses or other screens. We hope that this platform will serve as a tool to facilitate investigation of protein functions to the yeast community. © 2018 The Authors Yeast Published by John Wiley & Sons Ltd.

  2. Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora delbrueckii as model.

    PubMed

    Michel, Maximilian; Kopecká, Jana; Meier-Dörnberg, Tim; Zarnkow, Martin; Jacob, Fritz; Hutzler, Mathias

    2016-04-01

    This study describes a screening system for future brewing yeasts focusing on non-Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off-flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by-products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre-fermentation as a bio-flavouring agent for beers that have been post-fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour-forming properties. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast

    PubMed Central

    Zanders, Sarah E; Eickbush, Michael T; Yu, Jonathan S; Kang, Ji-Won; Fowler, Kyle R; Smith, Gerald R; Malik, Harmit Singh

    2014-01-01

    Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation. DOI: http://dx.doi.org/10.7554/eLife.02630.001 PMID:24963140

  4. Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast.

    PubMed

    Zanders, Sarah E; Eickbush, Michael T; Yu, Jonathan S; Kang, Ji-Won; Fowler, Kyle R; Smith, Gerald R; Malik, Harmit Singh

    2014-06-24

    Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation.DOI: http://dx.doi.org/10.7554/eLife.02630.001. Copyright © 2014, Zanders et al.

  5. Lager Yeast Comes of Age

    PubMed Central

    2014-01-01

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

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

    PubMed Central

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

  7. Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

    PubMed Central

    2013-01-01

    Background HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Results Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Conclusions Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I. PMID:24229420

  8. Protein–protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling.

    PubMed

    Ishii, Jun; Fukuda, Nobuo; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko

    2010-05-01

    For elucidating protein–protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available.Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins,and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.

  9. Yeast one-hybrid system used to identify the binding proteins for rat glutathione S-transferase P enhancer I.

    PubMed

    Liao, Ming-Xiang; Liu, Dong-Yuan; Zuo, Jin; Fang, Fu-De

    2002-03-01

    To detect the trans-factors specifically binding to the strong enhancer element (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. Yeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library to identify potential trans-factors that can interact with core sequence of GPEI(cGPEI). Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of transfactors to cGPEI. cDNA fragments coding for the C-terminal part of the transcription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. The binding of c-Jun and ANT to GPEI core sequence were confirmed. Rat c-jun transcriptional factor and ANT may interact with cGPEI. They could play an important role in the induced expression of GST-P gene.

  10. Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

    PubMed

    Zhang, Chenlu; Acosta-Sampson, Ligia; Yu, Vivian Yaci; Cate, Jamie H D

    2017-01-01

    The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

  11. New yeasts-new brews: modern approaches to brewing yeast design and development.

    PubMed

    Gibson, B; Geertman, J-M A; Hittinger, C T; Krogerus, K; Libkind, D; Louis, E J; Magalhães, F; Sampaio, J P

    2017-06-01

    The brewing industry is experiencing a period of change and experimentation largely driven by customer demand for product diversity. This has coincided with a greater appreciation of the role of yeast in determining the character of beer and the widespread availability of powerful tools for yeast research. Genome analysis in particular has helped clarify the processes leading to domestication of brewing yeast and has identified domestication signatures that may be exploited for further yeast development. The functional properties of non-conventional yeast (both Saccharomyces and non-Saccharomyces) are being assessed with a view to creating beers with new flavours as well as producing flavoursome non-alcoholic beers. The discovery of the psychrotolerant S. eubayanus has stimulated research on de novo S. cerevisiae × S. eubayanus hybrids for low-temperature lager brewing and has led to renewed interest in the functional importance of hybrid organisms and the mechanisms that determine hybrid genome function and stability. The greater diversity of yeast that can be applied in brewing, along with an improved understanding of yeasts' evolutionary history and biology, is expected to have a significant and direct impact on the brewing industry, with potential for improved brewing efficiency, product diversity and, above all, customer satisfaction. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Screening interspecific hybrids of Populus (P. ciliata x maximowiczii) using AFLP markers.

    PubMed

    Chauhan, N; Negi, M S; Sabharwal, V; Khurana, D K; Lakshmikumaran, M

    2004-03-01

    Hybrids of Populus ciliata x maximowiczii are very vigorous and outperform both the parents in growth performance and yield. Genetic evaluation of 24 of these interspecific hybrids along with the two mother trees ( Populus ciliata), and five male-parent ( Populus maximowiczii) genotypes was carried out using the AFLP marker assay. Eight AFLP primer combinations detected 428 markers, of which 280 (66%) were polymorphic. Genetic relationships within the samples were evaluated by generating the similarity matrix based on Jaccard's coefficient. The phenetic dendrograms, as well as the PCO plots, separated the hybrids and the two parent species into three distinct clusters. The hybrids grouped closer to the P. ciliata (female parent) cluster as compared to the P. maximowiczii (male parent) cluster. The hybrid cluster contained internal groupings, which correlated to some extent with growth performance. The four best performing hybrids (42m1, 65m1, 23m2, Cm2-5-20/91) formed a distinct sub-cluster. Data from a single primer combination was sufficient for distinguishing the hybrids from the parents and assigning paternity. The hybrids showed 22 markers that were absent in P. ciliata but were monomorphically present in all the hybrids, suggesting outcrossing and common paternity. Further, these 22 markers were found in all the P. maximowiczii genotypes confirming it as the male parent. These male-specific markers can be converted to SCAR markers and used for rapid screening of the P.ciliata x maximowiczii hybrids. The primer combination E-AAC x M-CAA was identified as most suitable for ascertaining true hybridity. AFLP proves to be a useful tool for screening of P. ciliata x maximowiczii hybrids at the early stages of development.

  13. Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609.

    PubMed

    Sambles, Christine; Middelhaufe, Sabine; Soanes, Darren; Kolak, Dagmara; Lux, Thomas; Moore, Karen; Matoušková, Petra; Parker, David; Lee, Rob; Love, John; Aves, Stephen J

    2017-09-01

    Most eukaryotic oleaginous species are yeasts and among them the basidiomycete red yeast, Rhodotorula ( Rhodosporidium ) toruloides (Pucciniomycotina) is known to produce high quantities of lipids when grown in nitrogen-limiting media, and has potential for biodiesel production. The genome of the CGMCC 2.1609 strain of this oleaginous red yeast was sequenced using a hybrid of Roche 454 and Illumina technology generating 13 × coverage. The de novo assembly was carried out using MIRA and scaffolded using MAQ and BAMBUS. The sequencing and assembly resulted in 365 scaffolds with total genome size of 33.4 Mb. The complete genome sequence of this strain was deposited in GenBank and the accession number is LKER00000000. The annotation is available on Figshare (doi:10.6084/m9.figshare.4754251).

  14. Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

    PubMed

    Chen, Jun; Young, Susan M; Allen, Chris; Seeber, Andrew; Péli-Gulli, Marie-Pierre; Panchaud, Nicolas; Waller, Anna; Ursu, Oleg; Yao, Tuanli; Golden, Jennifer E; Strouse, J Jacob; Carter, Mark B; Kang, Huining; Bologa, Cristian G; Foutz, Terry D; Edwards, Bruce S; Peterson, Blake R; Aubé, Jeffrey; Werner-Washburne, Margaret; Loewith, Robbie J; De Virgilio, Claudio; Sklar, Larry A

    2012-04-20

    TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.

  15. Metabolomics-based prediction models of yeast strains for screening of metabolites contributing to ethanol stress tolerance

    NASA Astrophysics Data System (ADS)

    Hashim, Z.; Fukusaki, E.

    2016-06-01

    The increased demand for clean, sustainable and renewable energy resources has driven the development of various microbial systems to produce biofuels. One of such systems is the ethanol-producing yeast. Although yeast produces ethanol naturally using its native pathways, production yield is low and requires improvement for commercial biofuel production. Moreover, ethanol is toxic to yeast and thus ethanol tolerance should be improved to further enhance ethanol production. In this study, we employed metabolomics-based strategy using 30 single-gene deleted yeast strains to construct multivariate models for ethanol tolerance and screen metabolites that relate to ethanol sensitivity/tolerance. The information obtained from this study can be used as an input for strain improvement via metabolic engineering.

  16. Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

    PubMed

    Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

    2014-09-01

    Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A Screened Hybrid DFT Study of Actinide Oxides, Nitrides, and Carbides

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wen, Xiaodong; Martin, Richard L.; Scuseria, Gustavo E.

    2013-06-27

    A systematic study of the structural, electronic, and magnetic properties of actinide oxides, nitrides, and carbides (AnX1–2 with X = C, N, O) is performed using the Heyd–Scuseria–Ernzerhof (HSE) hybrid functional. Our computed results show that the screened hybrid HSE functional gives a good description of the electronic and structural properties of actinide dioxides (strongly correlated insulators) when compared with available experimental data. However, there are still some problems reproducing the electronic properties of actinide nitrides and carbides (strongly correlated metals). In addition, in order to compare with the results by HSE, the structures, electronic, and magnetic properties of thesemore » actinide compounds are also investigated in the PBE and PBE+U approximation. Interestingly, the density of states of UN obtained with PBE compares well with the experimental photoemission spectra, in contrast to the hybrid approximation. This is presumably related to the need of additional screening in the Hartree–Fock exchange term of the metallic phases.« less

  18. Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking.

    PubMed

    Magalhães, Frederico; Krogerus, Kristoffer; Castillo, Sandra; Ortiz-Julien, Anne; Dequin, Sylvie; Gibson, Brian

    2017-08-01

    Yeast cryotolerance brings some advantages for wine fermentations, including the improved aromatic complexity of white wines. Naturally cold-tolerant strains are generally less adept at wine fermentation but fermentative fitness can potentially be improved through hybridization. Here we studied the potential of using hybrids involving Saccharomyces eubayanus and a S. cerevisiae wine strain for low-temperature winemaking. Through screening the performance in response to variable concentrations of sugar, nitrogen and temperature, we isolated one hybrid strain that exhibited the superior performance. This hybrid strain was propagated and dried in pilot scale and tested for the fermentation of Macabeu and Sauvignon blanc grape musts. We obtained highly viable active dry yeast, which was able to efficiently ferment the grape musts with superior production of aroma active volatiles, in particular, 2-phenylethanol. The genome sequences of the hybrid strains revealed variable chromosome inheritance among hybrids, particularly within the S. cerevisiae subgenome. With the present paper, we expand the knowledge on the potentialities of using S. eubayanus hybrids in industrial fermentation at beverages other than lager beer. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

    PubMed

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

    2015-08-25

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  20. Screening wild yeast strains for alcohol fermentation from various fruits.

    PubMed

    Lee, Yeon-Ju; Choi, Yu-Ri; Lee, So-Young; Park, Jong-Tae; Shim, Jae-Hoon; Park, Kwan-Hwa; Kim, Jung-Wan

    2011-03-01

    Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five α-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the α-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except α-amylase production, and fermented alcohol better than commercial wine yeasts.

  1. Complex Ancestries of Lager-Brewing Hybrids Were Shaped by Standing Variation in the Wild Yeast Saccharomyces eubayanus.

    PubMed

    Peris, David; Langdon, Quinn K; Moriarty, Ryan V; Sylvester, Kayla; Bontrager, Martin; Charron, Guillaume; Leducq, Jean-Baptiste; Landry, Christian R; Libkind, Diego; Hittinger, Chris Todd

    2016-07-01

    Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike ale-style beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains to each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. We conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.

  2. Complex ancestries of lager-brewing hybrids were shaped by standing variation in the wild yeast Saccharomyces eubayanus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peris, David; Langdon, Quinn K.; Moriarty, Ryan V.

    Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike alestyle beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains tomore » each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. Furthermore, we conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.« less

  3. Complex Ancestries of Lager-Brewing Hybrids Were Shaped by Standing Variation in the Wild Yeast Saccharomyces eubayanus

    PubMed Central

    Sylvester, Kayla; Charron, Guillaume; Leducq, Jean-Baptiste; Landry, Christian R.; Libkind, Diego; Hittinger, Chris Todd

    2016-01-01

    Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike ale-style beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains to each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. We conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes. PMID:27385107

  4. Complex ancestries of lager-brewing hybrids were shaped by standing variation in the wild yeast Saccharomyces eubayanus

    DOE PAGES

    Peris, David; Langdon, Quinn K.; Moriarty, Ryan V.; ...

    2016-07-06

    Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike alestyle beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains tomore » each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. Furthermore, we conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.« less

  5. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

    PubMed

    Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

    2008-02-01

    The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

  6. Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance.

    PubMed

    Weckesser, S; Engel, K; Simon-Haarhaus, B; Wittmer, A; Pelz, K; Schempp, C M

    2007-08-01

    There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.

  7. High-throughput analysis of the protein sequence-stability landscape using a quantitative "yeast surface two-hybrid" system and fragment reconstitution

    PubMed Central

    Dutta, Sanjib; Koide, Akiko; Koide, Shohei

    2008-01-01

    Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore the sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a "yeast surface two-hybrid" method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are predominantly recovered. Thus, it allows for the isolation of sequences that exhibits a desired level of stability. We identified over one hundred unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means to rapidly assess stability of many variants, to systematically assess contribution of different factors to protein stability and to enhance protein stability. PMID:18674545

  8. Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

    PubMed

    Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

    2018-02-08

    Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

  9. 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.

  10. Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.

    PubMed

    Urcuqui-Inchima, S; Walter, J; Drugeon, G; German-Retana, S; Haenni, A L; Candresse, T; Bernardi, F; Le Gall, O

    1999-05-25

    Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro. Copyright 1999 Academic Press.

  12. Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

    PubMed

    Kucejová, B; Foury, F

    2003-01-01

    RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

  13. Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

    PubMed Central

    McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

    1993-01-01

    Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

  14. Where Have All the Interactions Gone? Estimating the Coverage of Two-Hybrid Protein Interaction Maps

    PubMed Central

    Huang, Hailiang; Jedynak, Bruno M; Bader, Joel S

    2007-01-01

    Yeast two-hybrid screens are an important method for mapping pairwise physical interactions between proteins. The fraction of interactions detected in independent screens can be very small, and an outstanding challenge is to determine the reason for the low overlap. Low overlap can arise from either a high false-discovery rate (interaction sets have low overlap because each set is contaminated by a large number of stochastic false-positive interactions) or a high false-negative rate (interaction sets have low overlap because each misses many true interactions). We extend capture–recapture theory to provide the first unified model for false-positive and false-negative rates for two-hybrid screens. Analysis of yeast, worm, and fly data indicates that 25% to 45% of the reported interactions are likely false positives. Membrane proteins have higher false-discovery rates on average, and signal transduction proteins have lower rates. The overall false-negative rate ranges from 75% for worm to 90% for fly, which arises from a roughly 50% false-negative rate due to statistical undersampling and a 55% to 85% false-negative rate due to proteins that appear to be systematically lost from the assays. Finally, statistical model selection conclusively rejects the Erdös-Rényi network model in favor of the power law model for yeast and the truncated power law for worm and fly degree distributions. Much as genome sequencing coverage estimates were essential for planning the human genome sequencing project, the coverage estimates developed here will be valuable for guiding future proteomic screens. All software and datasets are available in Datasets S1 and S2, Figures S1–S5, and Tables S1−S6, and are also available from our Web site, http://www.baderzone.org. PMID:18039026

  15. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

  16. Screened hybrid density functionals for solid-state chemistry and physics.

    PubMed

    Janesko, Benjamin G; Henderson, Thomas M; Scuseria, Gustavo E

    2009-01-21

    Density functional theory incorporating hybrid exchange-correlation functionals has been extraordinarily successful in providing accurate, computationally tractable treatments of molecular properties. However, conventional hybrid functionals can be problematic for solids. Their nonlocal, Hartree-Fock-like exchange term decays slowly and incorporates unphysical features in metals and narrow-bandgap semiconductors. This article provides an overview of our group's work on designing hybrid functionals for solids. We focus on the Heyd-Scuseria-Ernzerhof screened hybrid functional [J. Chem. Phys. 2003, 118, 8207], its applications to the chemistry and physics of solids and surfaces, and our efforts to build upon its successes.

  17. Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1.

    PubMed Central

    Kalenik, J L; Chen, D; Bradley, M E; Chen, S J; Lee, T C

    1997-01-01

    Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation. To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, we screened an adult human muscle cDNA library using the yeast two-hybrid cloning system. We report the isolation and characterization of a novel protein termed YAF2 (YY1- associated factor 2) that interacts with YY1. The YAF2 cDNA encodes a 180 amino acid basic protein (pI 10.5) containing a single N-terminal C2-X10-C2 zinc finger. Lysine clusters are present that may function as a nuclear localization signal. Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction. In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells. YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1. Co-transfection of YY1 reverses the stimulatory effect of YAF2. YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium- activated protease m-calpain. The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development. PMID:9016636

  18. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

    PubMed

    Abécassis, V; Pompon, D; Truan, G

    2000-10-15

    The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

  19. Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

    PubMed Central

    Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

    2013-01-01

    The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

  20. Multiple Origins of the Pathogenic Yeast Candida orthopsilosis by Separate Hybridizations between Two Parental Species.

    PubMed

    Schröder, Markus S; Martinez de San Vicente, Kontxi; Prandini, Tâmara H R; Hammel, Stephen; Higgins, Desmond G; Bagagli, Eduardo; Wolfe, Kenneth H; Butler, Geraldine

    2016-11-01

    Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.

  1. N-Screen Aware Multicriteria Hybrid Recommender System Using Weight Based Subspace Clustering

    PubMed Central

    Ullah, Farman; Lee, Sungchang

    2014-01-01

    This paper presents a recommender system for N-screen services in which users have multiple devices with different capabilities. In N-screen services, a user can use various devices in different locations and time and can change a device while the service is running. N-screen aware recommendation seeks to improve the user experience with recommended content by considering the user N-screen device attributes such as screen resolution, media codec, remaining battery time, and access network and the user temporal usage pattern information that are not considered in existing recommender systems. For N-screen aware recommendation support, this work introduces a user device profile collaboration agent, manager, and N-screen control server to acquire and manage the user N-screen devices profile. Furthermore, a multicriteria hybrid framework is suggested that incorporates the N-screen devices information with user preferences and demographics. In addition, we propose an individual feature and subspace weight based clustering (IFSWC) to assign different weights to each subspace and each feature within a subspace in the hybrid framework. The proposed system improves the accuracy, precision, scalability, sparsity, and cold start issues. The simulation results demonstrate the effectiveness and prove the aforementioned statements. PMID:25152921

  2. The primary structures of two yeast enolase genes. Homology between the 5' noncoding flanking regions of yeast enolase and glyceraldehyde-3-phosphate dehydrogenase genes.

    PubMed

    Holland, M J; Holland, J P; Thill, G P; Jackson, K A

    1981-02-10

    Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5

  3. Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

    PubMed Central

    Xie, Rongrong; Zhou, Feng; Huang, Miao

    2017-01-01

    The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

  4. Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

    PubMed Central

    Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

    2010-01-01

    We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

  5. Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata.

    PubMed

    Gao, Jin-Xin; Jing, Jing; Yu, Chuan-Jin; Chen, Jie

    2015-06-01

    Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×10(5) transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×10(8) cfu/mL. The numbers for the cDNA library was 2.46×10(5). Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

  6. Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

    PubMed

    Garcia Sanchez, Rosa; Solodovnikova, Natalia; Wendland, Jürgen

    2012-08-01

    Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.

  7. 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

  8. Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking.

    PubMed

    Shohdy, Nadim; Efe, Jem A; Emr, Scott D; Shuman, Howard A

    2005-03-29

    Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

  9. Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts.

    PubMed

    Carrasco, Mario; Villarreal, Pablo; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

    2016-02-19

    Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases. All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi. Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting

  10. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    PubMed

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  11. [Effectiveness of FTA Elute® indicating cartridge in combination with hybrid capture 2 for cervical cancer screening].

    PubMed

    Chen, Feng; Zhang, Xi; Wang, Shaoming; Hu, Shangying; Chen, Wen; Zhao, Fanghui; He, Wei; Zhang, Yuqing; Qiao, Youlin

    2015-02-01

    To evaluate the effectiveness of FTA Elute® Cartridge (GE healthcare, Kent, UK) in combination with hybrid capture 2 (HC2) testing for cervical cancer screening. From May to June 2012, 412 women aged 25 to 65 years in Jiangxi Tonggu were enrolled in the study. We used pathological outcome as the gold standard, and the accuracy of the FTA card in combination with HC2 testing was investigated from both physician- and self-sampling, respectively. Physician sampling using the FTA card in combination with HC2 testing showed a comparable sensitivity (12/13) with the liquid based medium, but a higher specificity 69.5% (266/383) vs (77.8%, 298/383) (P < 0.001).When self sampling method was used, the sensitivity and specificity of using the FTA card in combination with HC2 testing with liquid based medium was 10/13 vs 8/13(P = 0.625) and (62.3%, 238/382) vs (75.7%, 289/382) (P < 0.001). The agreement of detection results for HC2 between FTA and liquid-based sampling medium was 86.1% (340/395) and 79.5% (314/395). For physician-collected samples used for HC2 testing to detect CIN2+, the accuracy of the FTA card was superior to that of the liquid-based medium (area under the receiver operating characteristic curve (AUC) = 0.898, 95%CI:0.838-0.958). FTA Elute® cartridge in combination with HC2 testing is a promising method of specimen transport for cervical cancer screening programs with a good precision.With further optimization, it could become an effective method for cervical cancer screening in various economic levels of areas.

  12. Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kita, Ayako; Higa, Mari; Doi, Akira

    Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis withmore » multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.« less

  13. Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease.

    PubMed

    Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Alvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

    2012-11-01

    MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  14. Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

    PubMed Central

    Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Álvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

    2012-01-01

    BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. PMID:22747838

  15. Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay.

    PubMed

    Heintz-Buschart, Anna; Eickhoff, Holger; Hohn, Erwin; Bilitewski, Ursula

    2013-03-10

    Candida albicans is one of the most common opportunistic fungal pathogens, causing life-threatening disease in immunocompromised patients. As it is not primarily a pathogen, but can exist in a commensal state, we aimed at the identification of new anti-infective compounds which do not eradicate the fungus, but primarily disable a virulence determinant. The yeast–hyphae-dimorphism of C. albicans is considered a major contributor to fungal disease, as mutants locked into either yeast or hyphal state have been shown to be less virulent in the mouse-model. We devised a high-throughput screening procedure which allows us to find inhibitors of the induction of hyphae. Hyphae-formation was induced by nitrogen starvation at 37 °C and neutral pH in a reporter strain, which couples promoter activity of the hyphae-specific HWP1 to β-galactosidase expression. In a pilot screening of 720 novel synthetic compounds, we identified substances which inhibited the outgrowth of germ tubes. They belonged to chemical classes not yet known for antimycotic properties, namely methyl aryl-oxazoline carboxylates, dihydrobenzo[d]isoxazolones and thiazolo[4,5-e]benzoisoxazoles. In conclusion we developed a novel screening assay, which addresses the morphological switch from the yeast form of C. albicans to its hyphal form and identified novel chemical structures with activity against C. albicans. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Editor’s Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast

    PubMed Central

    Lee, Sook-Jeong; Lee, Minho; Nam, Miyoung; Lee, Sol; Choi, Jian; Lee, Hye-Jin; Kim, Dong-Uk; Hoe, Kwang-Lae

    2018-01-01

    Abstract To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line. PMID:29294138

  17. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    USDA-ARS?s Scientific Manuscript database

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  18. Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

    PubMed

    Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

    2005-01-01

    We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

  19. Yeast as a tool to identify anti-aging compounds

    PubMed Central

    Zimmermann, Andreas; Hofer, Sebastian; Pendl, Tobias; Kainz, Katharina; Madeo, Frank; Carmona-Gutierrez, Didac

    2018-01-01

    Abstract In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae, has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification. PMID:29905792

  20. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    PubMed

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-04-22

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Screening mechanisms in hybrid metric-Palatini gravity

    NASA Astrophysics Data System (ADS)

    Santos, Marcelo Vargas dos; Alcaniz, Jailson S.; Mota, David F.; Capozziello, Salvatore

    2018-05-01

    We investigate the efficiency of screening mechanisms in the hybrid metric-Palatini gravity. The value of the field is computed around spherical bodies embedded in a background of constant density. We find a thin shell condition for the field depending on the background field value. To quantify how the thin shell effect is relevant, we analyze how it behaves in the neighborhood of different astrophysical objects (planets, moons, or stars). We find that the condition is very well satisfied except only for some peculiar objects. Furthermore we establish bounds on the model using data from Solar System experiments such as the spectral deviation measured by the Cassini mission and the stability of the Earth-Moon system, which gives the best constraint to date on f (R ) theories. These bounds contribute to fix the range of viable hybrid gravity models.

  2. A Novel Hybrid Iron Regulation Network Combines Features from Pathogenic and Nonpathogenic Yeasts.

    PubMed

    Gerwien, Franziska; Safyan, Abu; Wisgott, Stephanie; Hille, Fabrice; Kaemmer, Philipp; Linde, Jörg; Brunke, Sascha; Kasper, Lydia; Hube, Bernhard

    2016-10-18

    Iron is an essential micronutrient for both pathogens and their hosts, which restrict iron availability during infections in an effort to prevent microbial growth. Successful human pathogens like the yeast Candida glabrata have thus developed effective iron acquisition strategies. Their regulation has been investigated well for some pathogenic fungi and in the model organism Saccharomyces cerevisiae, which employs an evolutionarily derived system. Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi. Specifically, similarly to baker's yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes. However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens. Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi. The fungus Candida glabrata represents an evolutionarily close relative of the well-studied and benign baker's yeast and model organism Saccharomyces cerevisiae On the other hand, C. glabrata is an important opportunistic human pathogen causing both superficial and systemic infections. The ability to acquire trace metals, in particular, iron, and to tightly regulate this process during infection is considered an important virulence attribute of a variety of pathogens. Importantly, S. cerevisiae uses a highly derivative regulatory system distinct from those of other fungi. Until now, the regulatory mechanism of iron homeostasis in C. glabrata has been mostly unknown. Our study revealed a hybrid iron regulation network that is unique to C. glabrata and is placed at an evolutionary midpoint between those of S. cerevisiae and related fungal pathogens. We thereby

  3. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    PubMed

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  4. Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA

    PubMed Central

    Hellmuth, Klaus; Lau, Denise M.; Bischoff, F. Ralf; Künzler, Markus; Hurt, Ed; Simos, George

    1998-01-01

    Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-β-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast. PMID:9774653

  5. Alcoholic Fermentation of d-Xylose by Yeasts

    PubMed Central

    Toivola, Ansa; Yarrow, David; van den Bosch, Eduard; van Dijken, Johannes P.; Scheffers, W. Alexander

    1984-01-01

    Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used. PMID:16346558

  6. Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

    PubMed

    Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

    2017-01-31

    Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement

  7. Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2).

    PubMed

    Butler, Georgina S; Connor, Andrea R; Sounni, Nor Eddine; Eckhard, Ulrich; Morrison, Charlotte J; Noël, Agnès; Overall, Christopher M

    2017-05-01

    Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 "inactive catalytic domain capture" (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro-CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis. We find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues. Copyright © 2016 The Authors. Published by Elsevier

  8. 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.

  9. QTL mapping of sake brewing characteristics of yeast.

    PubMed

    Katou, Taku; Namise, Masahiro; Kitagaki, Hiroshi; Akao, Takeshi; Shimoi, Hitoshi

    2009-04-01

    A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.

  10. 21 CFR 172.381 - Vitamin D2 bakers yeast.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Vitamin D2 bakers yeast. 172.381 Section 172.381... CONSUMPTION Special Dietary and Nutritional Additives § 172.381 Vitamin D2 bakers yeast. Vitamin D2 bakers yeast may be used safely in foods as a source of vitamin D2 and as a leavening agent in accordance with...

  11. Improved microarray methods for profiling the yeast knockout strain collection

    PubMed Central

    Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

    2005-01-01

    A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

  12. Alternative Saccharomyces interspecies hybrid combinations and their potential for low‐temperature wort fermentation

    PubMed Central

    Nikulin, Jarkko; Krogerus, Kristoffer

    2017-01-01

    Abstract The lager yeast hybrid (Saccharomyces cerevisiae × Saccharomyces eubayanus) possesses two key characteristics that are essential for lager brewing: efficient sugar utilization and cold tolerance. Here we explore the possibility that the lager yeast phenotype can be recreated by hybridizing S. cerevisiae ale yeast with a number of cold‐tolerant Saccharomyces species including Saccharomyces arboricola, Saccharomyces eubayanus, Saccharomyces mikatae and Saccharomyces uvarum. Interspecies hybrids performed better than parental strains in lager brewing conditions (12°C and 12°P wort), with the S. mikatae hybrid performing as well as the S. eubayanus hybrid. Where the S. cerevisiae parent was capable of utilizing maltotriose, this trait was inherited by the hybrids. A greater production of higher alcohols and esters by the hybrids resulted in the production of more aromatic beers relative to the parents. Strong fermentation performance relative to the parents was dependent on ploidy, with polyploid hybrids (3n, 4n) performing better than diploid hybrids. All hybrids produced 4‐vinyl guaiacol, a smoke/clove aroma generally considered an off flavour in lager beer. This characteristic could however be eliminated by isolating spore clones from a fertile hybrid of S. cerevisiae and S. mikatae. The results suggest that S. eubayanus is dispensable when constructing yeast hybrids that express the typical lager yeast phenotype. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:28755430

  13. 21 CFR 172.381 - Vitamin D2 bakers yeast.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... conventional bakers yeast. (c) The additive may be used in yeast-leavened baked goods and baking mixes and yeast-leavened baked snack foods at levels not to exceed 400 International Units of vitamin D2 per 100...

  14. Performance study of sugar-yeast-ethanol bio-hybrid fuel cells

    NASA Astrophysics Data System (ADS)

    Jahnke, Justin P.; Mackie, David M.; Benyamin, Marcus; Ganguli, Rahul; Sumner, James J.

    2015-05-01

    Renewable alternatives to fossil hydrocarbons for energy generation are of general interest for a variety of political, economic, environmental, and practical reasons. In particular, energy from biomass has many advantages, including safety, sustainability, and the ability to be scavenged from native ecosystems or from waste streams. Microbial fuel cells (MFCs) can take advantage of microorganism metabolism to efficiently use sugar and other biomolecules as fuel, but are limited by low power densities. In contrast, direct alcohol fuel cells (DAFCs) take advantage of proton exchange membranes (PEMs) to generate electricity from alcohols at much higher power densities. Here, we investigate a novel bio-hybrid fuel cell design prepared using commercial off-the-shelf DAFCs. In the bio-hybrid fuel cells, biomass such as sugar is fermented by yeast to ethanol, which can be used to fuel a DAFC. A separation membrane between the fermentation and the DAFC is used to purify the fermentate while avoiding any parasitic power losses. However, shifting the DAFCs from pure alcohol-water solutions to filtered fermented media introduces complications related to how the starting materials, fermentation byproducts, and DAFC waste products affect both the fermentation and the long-term DAFC performance. This study examines the impact of separation membrane pore size, fermentation/fuel cell byproducts, alcohol and salt concentrations, and load resistance on fuel cell performance. Under optimized conditions, the performance obtained is comparable to that of a similar DAFC run with a pure alcohol-water mixture. Additionally, the modified DAFC can provide useable amounts of power for weeks.

  15. Construction of high quality Gateway™ entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon.

    PubMed

    Cao, Shuanghe; Siriwardana, Chamindika L; Kumimoto, Roderick W; Holt, Ben F

    2011-05-19

    Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings), developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium) is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H) libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark) and long day (LD, 20/4-h light/dark) grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each) and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user-defined purposes and the derived Y2H libraries can be

  16. Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

    PubMed Central

    Dunn, Barbara; Paulish, Terry; Stanbery, Alison; Piotrowski, Jeff; Koniges, Gregory; Kroll, Evgueny; Louis, Edward J.; Liti, Gianni; Sherlock, Gavin; Rosenzweig, Frank

    2013-01-01

    Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5′ portion derives from S. cerevisiae and the 3′ portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in

  17. Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment

    PubMed Central

    Erny, C.; Raoult, P.; Alais, A.; Butterlin, G.; Delobel, P.; Matei-Radoi, F.; Casaregola, S.

    2012-01-01

    The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δμ)2 genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates. PMID:22344648

  18. Exploration for the Salinity Tolerance-Related Genes from Xero-Halophyte Atriplex canescens Exploiting Yeast Functional Screening System

    PubMed Central

    Li, Jingtao; Sun, Xinhua; Liu, Yanzhi; Wang, Xueliang; Zhang, Hao; Pan, Hongyu

    2017-01-01

    Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 105 transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens. PMID:29149055

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

    PubMed

    Codón, Antonio C; Rincón, Ana M; Moreno-Mateos, Miguel A; Delgado-Jarana, Jesús; Rey, Manuel; Limón, Carmen; Rosado, Ivan V; Cubero, Beatriz; Peñate, Xenia; Castrejón, Francisco; Benítez, Tahía

    2003-01-15

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

  20. Molecular architecture of the yeast Mediator complex

    PubMed Central

    Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo; Greenberg, Charles H; Bushnell, David A; Davis, Ralph; Burlingame, Alma L; Sali, Andrej; Kornberg, Roger D

    2015-01-01

    The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. DOI: http://dx.doi.org/10.7554/eLife.08719.001 PMID:26402457

  1. Designing and creating Saccharomyces interspecific hybrids for improved, industry relevant, phenotypes.

    PubMed

    Bellon, Jennifer R; Yang, Fei; Day, Martin P; Inglis, Debra L; Chambers, Paul J

    2015-10-01

    To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.

  2. Disposable electrochemical immunosensor for Brettanomyces bruxellensis based on nanogold-reduced graphene oxide hybrid nanomaterial.

    PubMed

    Borisova, Boryana; Villalonga, María L; Arévalo-Villena, María; Boujakhrout, Abderrahmane; Sánchez, Alfredo; Parrado, Concepción; Pingarrón, José M; Briones-Pérez, Ana; Villalonga, Reynaldo

    2017-09-01

    The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10-10 6  CFU/mL and 10 2 -10 6  CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability. Graphical abstract A novel disposable electrochemical immunosensor for the detection of the red wine spoilage yeast B. bruxellensis.

  3. The genome sequence of Saccharomyces eubayanus and the domestication of lager-brewing yeasts

    DOE PAGES

    Baker, Emily Clare; Wang, Bing; Bellora, Nicolas; ...

    2015-08-11

    The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae ( S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts havemore » experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. In conclusion, our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions.« less

  4. The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts

    PubMed Central

    Baker, EmilyClare; Wang, Bing; Bellora, Nicolas; Peris, David; Hulfachor, Amanda Beth; Koshalek, Justin A.; Adams, Marie; Libkind, Diego; Hittinger, Chris Todd

    2015-01-01

    The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae (S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts have experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. Our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions. PMID:26269586

  5. Urine Multi-drug Screening with GC-MS or LC-MS-MS Using SALLE-hybrid PPT/SPE.

    PubMed

    Lee, Junhui; Park, Jiwon; Go, Ahra; Moon, Heesung; Kim, Sujin; Jung, Sohee; Jeong, Wonjoon; Chung, Heesun

    2018-05-14

    To intoxicated patients in the emergency room, toxicological analysis can be considerably helpful for identifying the involved toxicants. In order to develop a urine multi-drug screening (UmDS) method, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) were used to determine targeted and unknown toxicants in urine. A GC-MS method in scan mode was validated for selectivity, limit of detection (LOD) and recovery. An LC-MS-MS multiple reaction monitoring (MRM) method was validated for lower LOD, recovery and matrix effect. The results of the screening analysis were compared with patient medical records to check the reliability of the screen. Urine samples collected from an emergency room were extracted through a combination of salting-out assisted liquid-liquid extraction (SALLE) and hybrid protein precipitation/solid phase extraction (hybrid PPT/SPE) plates and examined by GC-MS and LC-MS-MS. GC-MS analysis was performed as unknown drug screen and LC-MS-MS analysis was conducted as targeted drug screen. After analysis by GC-MS, a library search was conducted using an in-house library established with the automated mass spectral deconvolution and identification system (AMDISTM). LC-MS-MS used Cliquid®2.0 software for data processing and acquisition in MRM mode. An UmDS method by GC-MS and LC-MS-MS was developed by using a SALLE-hybrid PPT/SPE and in-house library. The results of UmDS by GC-MS and LC-MS-MS showed that toxicants could be identified from 185 emergency room patient samples containing unknown toxicants. Zolpidem, acetaminophen and citalopram were the most frequently encountered drugs in emergency room patients. The UmDS analysis developed in this study can be used effectively to detect toxic substances in a short time. Hence, it could be utilized in clinical and forensic toxicology practices.

  6. An aminoacylation-dependent nuclear tRNA export pathway in yeast.

    PubMed

    Grosshans, H; Hurt, E; Simos, G

    2000-04-01

    Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

  7. An aminoacylation-dependent nuclear tRNA export pathway in yeast

    PubMed Central

    Grosshans, Helge; Hurt, Ed; Simos, George

    2000-01-01

    Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries. PMID:10766739

  8. Mitochondria inheritance is a key factor for tolerance to dehydration in wine yeast production.

    PubMed

    Picazo, C; Gamero-Sandemetrio, E; Orozco, H; Albertin, W; Marullo, P; Matallana, E; Aranda, A

    2015-03-01

    Mitochondria are the cell's powerhouse when organisms are grown in the presence of oxygen. They are also the source of reactive oxygen species that cause damage to the biochemical components of the cell and lead to cellular ageing and death. Under winemaking conditions, Saccharomyces yeasts exclusively have a fermentative metabolism due to the high sugar content of grape must. However, their production as an active dry yeast (ADY) form required aerobic propagation and a dehydration process. In these industrial steps, oxidative stress is particularly harmful for the cell. In this work, we analysed the impact of the mitochondrial genome on oxidative stress response, longevity and dehydration tolerance using the synthetic interspecific hybrids obtained between two S. cerevisiae and S. uvarum strains. The isogenic nature of nuclear DNA of such hybrids allowed us to analyse the impact of mitochondrial DNA for fermentative and oxidative stress conditions. Under grape must conditions, the inheritance of mitochondrial DNA poorly impacted the fermentative performance of interspecific hybrids, unlike the hybrids with S. cerevisiae mitochondrial inheritance, which displayed increased tolerance to oxidative stress and dehydration, and showed an extended chronological longevity when cells were grown with aeration. In modern oenology, yeast starters are employed to inoculate grape juice, usually in the form of active dry yeast (ADY). The dehydration process implies stressful conditions that lead to oxidative damage. Other yeast species and interspecific hybrids other than Saccharomyces cerevisiae may be used to confer novel properties to the final product. However, these yeasts are usually more sensitive to drying. Understanding the causes of oxidative stress tolerance is therefore necessary for developing the use of these organisms in industry. This study indicates the impact of mitochondrial DNA inheritance for oxidative stress resistance in an interspecific context using

  9. Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays.

    PubMed

    Martins, Flaviano S; Miranda, Ieso C; Rosa, Carlos A; Nicoli, Jacques R; Neves, Maria J

    2008-01-01

    Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

  10. Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii.

    PubMed

    Xu, Jianping; Yan, Zhun; Guo, Hong

    2009-06-01

    The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii. The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii, but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.

  11. Health benefits and cost-effectiveness of a hybrid screening strategy for colorectal cancer.

    PubMed

    Dinh, Tuan; Ladabaum, Uri; Alperin, Peter; Caldwell, Cindy; Smith, Robert; Levin, Theodore R

    2013-09-01

    Colorectal cancer (CRC) screening guidelines recommend screening schedules for each single type of test except for concurrent sigmoidoscopy and fecal occult blood test (FOBT). We investigated the cost-effectiveness of a hybrid screening strategy that was based on a fecal immunological test (FIT) and colonoscopy. We conducted a cost-effectiveness analysis by using the Archimedes Model to evaluate the effects of different CRC screening strategies on health outcomes and costs related to CRC in a population that represents members of Kaiser Permanente Northern California. The Archimedes Model is a large-scale simulation of human physiology, diseases, interventions, and health care systems. The CRC submodel in the Archimedes Model was derived from public databases, published epidemiologic studies, and clinical trials. A hybrid screening strategy led to substantial reductions in CRC incidence and mortality, gains in quality-adjusted life years (QALYs), and reductions in costs, comparable with those of the best single-test strategies. Screening by annual FIT of patients 50-65 years old and then a single colonoscopy when they were 66 years old (FIT/COLOx1) reduced CRC incidence by 72% and gained 110 QALYs for every 1000 people during a period of 30 years, compared with no screening. Compared with annual FIT, FIT/COLOx1 gained 1400 QALYs/100,000 persons at an incremental cost of $9700/QALY gained and required 55% fewer FITs. Compared with FIT/COLOx1, colonoscopy at 10-year intervals gained 500 QALYs/100,000 at an incremental cost of $35,100/QALY gained but required 37% more colonoscopies. Over the ranges of parameters examined, the cost-effectiveness of hybrid screening strategies was slightly more sensitive to the adherence rate with colonoscopy than the adherence rate with yearly FIT. Uncertainties associated with estimates of FIT performance within a program setting and sensitivities for flat and right-sided lesions are expected to have significant impacts on the cost

  12. High-performance TiO(2) from Baker's yeast.

    PubMed

    He, Wen; Cui, Jingjie; Yue, Yuanzheng; Zhang, Xudong; Xia, Xi; Liu, Hong; Lui, Suwen

    2011-02-01

    Based on the biomineralization assembly concept, a biomimetic approach has been developed to synthesize high-performance mesoporous TiO(2). The key step of this approach is to apply Baker's yeast cells as biotemplates for deriving the hierarchically ordered mesoporous anatase structure. The mechanism of formation of the yeast-TiO(2) is revealed by characterizing its morphology, microstructure, and chemical composition. The yeast-TiO(2) exhibits outstanding photocatalytic performance. Under visible-light irradiation, the removal efficiency of chemical oxygen demand (COD) and color of the paper industry wastewater has reached 80.3% and nearly 100%, respectively. The approach may open new vistas for fabricating advanced mesoporous materials under ambient condition. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts.

    PubMed

    Baker, EmilyClare; Wang, Bing; Bellora, Nicolas; Peris, David; Hulfachor, Amanda Beth; Koshalek, Justin A; Adams, Marie; Libkind, Diego; Hittinger, Chris Todd

    2015-11-01

    The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae (S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts have experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. Our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. Large-Scale Selection and Breeding To Generate Industrial Yeasts with Superior Aroma Production

    PubMed Central

    Steensels, Jan; Meersman, Esther; Snoek, Tim; Saels, Veerle

    2014-01-01

    The concentrations and relative ratios of various aroma compounds produced by fermenting yeast cells are essential for the sensory quality of many fermented foods, including beer, bread, wine, and sake. Since the production of these aroma-active compounds varies highly among different yeast strains, careful selection of variants with optimal aromatic profiles is of crucial importance for a high-quality end product. This study evaluates the production of different aroma-active compounds in 301 different Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus yeast strains. Our results show that the production of key aroma compounds like isoamyl acetate and ethyl acetate varies by an order of magnitude between natural yeasts, with the concentrations of some compounds showing significant positive correlation, whereas others vary independently. Targeted hybridization of some of the best aroma-producing strains yielded 46 intraspecific hybrids, of which some show a distinct heterosis (hybrid vigor) effect and produce up to 45% more isoamyl acetate than the best parental strains while retaining their overall fermentation performance. Together, our results demonstrate the potential of large-scale outbreeding to obtain superior industrial yeasts that are directly applicable for commercial use. PMID:25192996

  15. Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

    PubMed Central

    Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

    2000-01-01

    A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

  16. Application of genetics to the development of starch-fermenting yeasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mattoon, J.R.; Kim, K.; Laluce, C.

    1987-01-01

    Yeast strains capable of direct fermentation of manioc starch were developed by hybridizing strains of Saccharomyces diastaticus and Saccharomyces cerevisiae. Hybrids were evaluated for speed of alcohol production, and yields and speed of formation of glycoamylase. Up to 6% solutions of Lintner starch could be fermented directly with about 80% conversion to alcohol. Pretreatment of crude 40% manioc starch suspensions with alpha-amylase, followed by fermentations with a starch-fermenting yeast strain, permitted accumulation of 12% ethanol within three days. Starch conversion was almost 100%. A fragment of DNA was cloned from S. diastaticus using the yeast-E. coli shuttle vector, YEp13, andmore » was used to transform a strain of S. cerevisiae to a starch-fermenting state. Supported by National Science Foundation grant INT 7927328 and National Institutes of Health grant GM 27860. Dr. Laluce was supported by a grant from Fundacao de Amparo a Pesquisa do Estado do Sao Paulo and by her university. (Refs. 5).« less

  17. Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70.

    PubMed

    Cousseau, F E M; Alves, S L; Trichez, D; Stambuk, B U

    2013-01-01

    The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort. © 2012 The Society for Applied Microbiology.

  18. Yeast for virus research

    PubMed Central

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  19. Development of 40-in hybrid hologram screen for auto-stereoscopic video display

    NASA Astrophysics Data System (ADS)

    Song, Hyun Ho; Nakashima, Y.; Momonoi, Y.; Honda, Toshio

    2004-06-01

    Usually in auto stereoscopic display, there are two problems. The first problem is that large image display is difficult, and the second problem is that the view zone (which means the zone in which both eyes are put for stereoscopic or 3-D image observation) is very narrow. We have been developing an auto stereoscopic large video display system (over 100 inches diagonal) which a few people can view simultaneously1,2. Usually in displays that are over 100 inches diagonal, an optical video projection system is used. As one of auto stereoscopic display systems the hologram screen has been proposed3,4,5,6. However, if the hologram screen becomes too large, the view zone (corresponding to the reconstructed diffused object) causes color dispersion and color aberration7. We also proposed the additional Fresnel lens attached to the hologram screen. We call the screen a "hybrid hologram screen", (HHS in short). We made the HHS 866mm(H)×433mm(V) (about 40 inch diagonal)8,9,10,11. By using the lens in the reconstruction step, the angle between object light and reference light can be small, compared to without the lens. So, the spread of the view zone by the color dispersion and color aberration becomes small. And also, the virtual image which is reconstructed from the hologram screen can be transformed to a real image (view zone). So, it is not necessary to use a large lens or concave mirror while making a large hologram screen.

  20. Bread, beer and wine: yeast domestication in the Saccharomyces sensu stricto complex.

    PubMed

    Sicard, Delphine; Legras, Jean-Luc

    2011-03-01

    Yeasts of the Saccharomyces sensu stricto species complex are able to convert sugar into ethanol and CO(2) via fermentation. They have been used for thousands years by mankind for fermenting food and beverages. In the Neolithic times, fermentations were probably initiated by naturally occurring yeasts, and it is unknown when humans started to consciously add selected yeast to make beer, wine or bread. Interestingly, such human activities gave rise to the creation of new species in the Saccharomyces sensu stricto complex by interspecies hybridization or polyploidization. Within the S. cerevisiae species, they have led to the differentiation of genetically distinct groups according to the food process origin. Although the evolutionary history of wine yeast populations has been well described, the histories of other domesticated yeasts need further investigation. Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  1. New Lager Brewery Strains Obtained by Crossing Techniques Using Cachaça (Brazilian Spirit) Yeasts

    PubMed Central

    Figueiredo, Bruna Inez Carvalho; Saraiva, Margarete Alice Fontes; de Souza Pimenta, Paloma Patrick; de Souza Testasicca, Miriam Conceição; Sampaio, Geraldo Magela Santos; da Cunha, Aureliano Claret; Afonso, Luis Carlos Crocco; Vieira de Queiroz, Marisa; de Miranda Castro, Ieso

    2017-01-01

    ABSTRACT The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach. IMPORTANCE Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in

  2. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    PubMed Central

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  3. A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A.

    PubMed

    Wu, Lin; Li, Huijun; Tang, Tianle

    2017-01-24

    Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence) protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21) and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris . Different expression conditions of sortase A include: induction temperature (22 °C, 28 °C, 37 °C and 40 °C), induction time (4 h, 5 h, 6 h and 7 h) and induction concentration of isopropyl β-d-thiogalactoside IPTG (0.25 mmol/L, 0.5 mmol/L, 1 mmol/L, and 2 mmol/L). The fluorescence change of the LPETG-EGFP protein on the surface of P. pastoris over time was detected by flow cytometry and fluorescence spectrophotometry, and then the sensitivities of the two methods were compared. Using berberine chloride as an inhibitor, the activity of sortase A was investigated with the substrates of LPETG-EGFP protein, and compared to Dabcyl-QALPETGEE-Edans. A high yield of sortase A was achieved by inducing 1.0 mmol/L IPTG at 28 °C for 6 h. The intensity of green fluorescence of substrates displayed on the yeast surface was increased over time, while the stability was decreased slightly. Both fluorescence spectrophotometery and flow cytometry were fit for detection because of their high sensitivity. We utilized two different substrates of sortase A to investigate sortase A activity, which resulted in the increase of fluorescence intensity with respect to the increased time of growth. However, the method with Dabcyl-QALPETGEE-Edans as its substrate was more robust. Thus, the method described in this paper is a simple and cheap method which is very suitable for

  4. A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

    PubMed Central

    Mertens, Stijn; Steensels, Jan; Saels, Veerle; De Rouck, Gert; Aerts, Guido

    2015-01-01

    Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, “Saaz” and “Frohberg.” This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma. PMID:26407881

  5. Inheritance of brewing-relevant phenotypes in constructed Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids.

    PubMed

    Krogerus, Kristoffer; Seppänen-Laakso, Tuulikki; Castillo, Sandra; Gibson, Brian

    2017-04-21

    Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity. Here we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn't produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose. Our study demonstrates the

  6. The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

    PubMed Central

    Wahba, Lamia; Gore, Steven K; Koshland, Douglas

    2013-01-01

    Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo. DOI: http://dx.doi.org/10.7554/eLife.00505.001 PMID:23795288

  7. Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids.

    PubMed

    Zuchowska, Magdalena; Jaenicke, Elmar; König, Helmut; Claus, Harald

    2015-11-01

    The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast. Copyright © 2015 John Wiley & Sons, Ltd.

  8. A large set of newly created interspecific Saccharomyces hybrids increases aromatic diversity in lager beers.

    PubMed

    Mertens, Stijn; Steensels, Jan; Saels, Veerle; De Rouck, Gert; Aerts, Guido; Verstrepen, Kevin J

    2015-12-01

    Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, "Saaz" and "Frohberg." This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Screening protocol for Torulopsis (Candida) glabrata.

    PubMed Central

    Land, G; Burke, J; Shelby, C; Rhodes, J; Collett, J; Bennett, I; Johnson, J

    1996-01-01

    A screening test has been developed for the presumptive identification of Torulopsis (Candida) glabrata from other common clinical isolates of yeast-like fungi. An interlaboratory comparison of a protocol consisting of morphology on cornmeal Tween 80 agar and trehalose fermentation at 42 degrees C was successful in differentiating T. glabrata from other taxa that are frequent or possible clinical isolates. The screening results for 517 clinical yeast isolates, 241 of which were T. glabrata, were compared with their final identification via commercial systems (API20C Yeast Identification System [bioMERIEUX, Hazelwood, Mo.] and Rapid Yeast Identification Panel [Dade Microscan, Sacramento, Calif.]). The trehalose screening test has a sensitivity and a specificity of 97.8 and 95.8%, respectively, and a positive predictive value of 97.4% and a negative predictive value of 96.5%. Overall, the trehalose screen had an efficiency rating of 93.9% for ruling in or out T. glabrata. Since T. glabrata represents a substantial part of the workload in a clinical laboratory, a significant reduction in direct and indirect costs should be realized. PMID:8862605

  10. Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

    PubMed Central

    Sliva, Anna; Kuang, Zheng; Meluh, Pamela B.; Boeke, Jef D.

    2016-01-01

    The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating. PMID:26837954

  11. Functional mapping of yeast genomes by saturated transposition

    PubMed Central

    Michel, Agnès H; Hatakeyama, Riko; Kimmig, Philipp; Arter, Meret; Peter, Matthias; Matos, Joao; De Virgilio, Claudio; Kornmann, Benoît

    2017-01-01

    Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput. DOI: http://dx.doi.org/10.7554/eLife.23570.001 PMID:28481201

  12. Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

    PubMed

    Cappello, M S; Poltronieri, P; Blaiotta, G; Zacheo, G

    2010-11-15

    The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner

    PubMed Central

    Allonso, Diego; Nogueira, Mauricio L.; Mohana-Borges, Ronaldo

    2013-01-01

    Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection. PMID:23516407

  14. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that anmore » rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.« less

  15. A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast

    PubMed Central

    Yadav, Akhilesh K.; Shankar, Alka; Jha, Saroj K.; Kanwar, Poonam; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA (calcium cation exchangers) superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca2+ starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca2+ sensitivity of K667 (low affinity Ca2+ uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na+, Li+, Fe2+, Zn2+ and Co2+ and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K+ in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil. PMID:26607171

  16. BAPJ69-4A: a yeast two-hybrid strain for both positive and negative genetic selection.

    PubMed

    Shaffer, Hally Anne; Rood, Michael Kenneth; Kashlan, Badar; Chang, Eileen I-ling; Doyle, Donald Francis; Azizi, Bahareh

    2012-10-01

    Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein-protein and protein-ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koopman, R P; Langlois, R G; Nasarabadi, S

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

  18. Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast

    PubMed Central

    Kelemen, Zsolt; Sebastian, Alvaro; Xu, Wenjia; Grain, Damaris; Salsac, Fabien; Avon, Alexandra; Berger, Nathalie; Tran, Joseph; Dubreucq, Bertrand; Lurin, Claire; Lepiniec, Loïc; Contreras-Moreira, Bruno; Dubos, Christian

    2015-01-01

    The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences. PMID:26484765

  19. Combinatorial high-throughput optical screening of high performance Pd alloy cathode for hybrid Li-air battery.

    PubMed

    Jun, Young Jin; Park, Sung Hyeon; Woo, Seong Ihl

    2014-12-08

    Combinatorial high-throughput optical screening method was developed to find the optimum composition of highly active Pd-based catalysts at the cathode of the hybrid Li-air battery. Pd alone, which is one-third the cost of Pt, has difficulty in replacing Pt; therefore, the integration of other metals was investigated to improve its performance toward oxygen reduction reaction (ORR). Among the binary Pd-based catalysts, the composition of Pd-Ir derived catalysts had higher performance toward ORR compared to other Pd-based binary combinations. The composition at 88:12 at. % (Pd: Ir) showed the highest activity toward ORR at the cathode of the hybrid Li-air battery. The prepared Pd(88)Ir(12)/C catalyst showed a current density of -2.58 mA cm(-2) at 0.8 V (vs RHE), which was around 30% higher compared to that of Pd/C (-1.97 mA cm(-2)). When the prepared Pd(88)Ir(12)/C catalyst was applied to the hybrid Li-air battery, the polarization of the cell was reduced and the energy efficiency of the cell was about 30% higher than that of the cell with Pd/C.

  20. Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes.

    PubMed

    Goldberg, Alexander A; Richard, Vincent R; Kyryakov, Pavlo; Bourque, Simon D; Beach, Adam; Burstein, Michelle T; Glebov, Anastasia; Koupaki, Olivia; Boukh-Viner, Tatiana; Gregg, Christopher; Juneau, Mylène; English, Ann M; Thomas, David Y; Titorenko, Vladimir I

    2010-07-01

    In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA.

  1. Rapid Parallel Screening for Strain Optimization

    DTIC Science & Technology

    2013-08-16

    fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can...reporter, and, 2) a yeast TAR cloning shuttle vector for transferring catabolic clusters to E. coli. 15. SUBJECT TERMS NA 16. SECURITY CLASSIFICATION OF... fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can utilize

  2. Rapid Parallel Screening for Strain Optimization

    DTIC Science & Technology

    2013-05-16

    fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can...reporter, and, 2) a yeast TAR cloning shuttle vector for transferring catabolic clusters to E. coli. 15. SUBJECT TERMS NA 16. SECURITY CLASSIFICATION OF... fermentation yields of industrially relevant biological compounds. Screening of the desired chemicals was completed previously. Microbes that can utilize

  3. Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses.

    PubMed

    Mohamed, Mohamed R; Rahman, Masmudur M; Lanchbury, Jerry S; Shattuck, Donna; Neff, Chris; Dufford, Max; van Buuren, Nick; Fagan, Katharine; Barry, Michele; Smith, Scott; Damon, Inger; McFadden, Grant

    2009-06-02

    Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-kappaB1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-kappaB1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-kappaB signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.

  4. Identification of superior lipid producing Lipomyces and Myxozyma yeasts

    USDA-ARS?s Scientific Manuscript database

    Oleaginous yeasts are of interest for production of single cell oils from sugars. Here 17 members of the Lipomyces and Myxozyma clade were screened for lipid production when cultured on glucose. The highest ranking yeasts included L. tetrasporus (21 g/l), L. kononenkoae (19.6 g/l), and L. lipofer (1...

  5. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Park, E.; Prakash, L.; Guzder, S.N.

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M[sub r] 95,356). Themore » RAD25 (SSL2)- and XPCX-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UN sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys[sup 392] residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD 25 (SSL2) ATPase/DNA helicase activity in viability. 40 refs., 3 figs., 1 tab.« less

  6. New family of pectinase genes PGU1b-PGU3b of the pectinolytic yeast Saccharomyces bayanus var. uvarum.

    PubMed

    Naumov, G I; Shalamitskiy, M Yu; Naumova, E S

    2016-03-01

    Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.

  7. Yeasts in sustainable bioethanol production: A review.

    PubMed

    Mohd Azhar, Siti Hajar; Abdulla, Rahmath; Jambo, Siti Azmah; Marbawi, Hartinie; Gansau, Jualang Azlan; Mohd Faik, Ainol Azifa; Rodrigues, Kenneth Francis

    2017-07-01

    Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.

  8. Accelerating Yeast Prion Biology using Droplet Microfluidics

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  9. CKA2 functions in H2O2-induced apoptosis and high-temperature stress tolerance by regulating NO accumulation in yeast.

    PubMed

    Liu, Wen-Cheng; Yuan, Hong-Mei; Li, Yun-Hui; Lu, Ying-Tang

    2015-09-01

    Nitric oxide (NO) plays key roles in yeast responses to various environmental factors, such as H2O2 and high temperature. However, the gene encoding NO synthase (NOS) in yeast has not yet been identified, and the mechanism underlying the regulation of NOS-like activity is poorly understood. Here, we report on the involvement of CKA2 in H2O2-induced yeast apoptosis and yeast high-temperature stress tolerance. Our results showed that although Δcka2 mutant had reduced NO accumulation with decreased apoptosis after H2O2 exposure, treatment with a NO donor, sodium nitroprusside, resulted in similar survival rate of Δcka2 mutant compared to that of wild-type yeast when subjected to H2O2 stress. This finding occurred because H2O2-enhanced NOS-like activity in wild-type yeast was significantly repressed in Δcka2. Our additional experiments indicated that both high-temperature-enhanced NO accumulation and NOS-like activity were also suppressed in Δcka2, leading to the hypersensitivity of the mutant to high temperature in terms of changes in survival rate. Thus, our results showed that CKA2 functioned in H2O2-induced apoptosis and high-temperature stress tolerance by regulating NOS-like-dependent NO accumulation in yeast. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

    PubMed

    Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

    2011-01-01

    cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

  11. A breeding strategy to harness flavor diversity of Saccharomyces interspecific hybrids and minimize hydrogen sulfide production.

    PubMed

    Bizaj, Etjen; Cordente, Antonio G; Bellon, Jennifer R; Raspor, Peter; Curtin, Chris D; Pretorius, Isak S

    2012-06-01

    Industrial food-grade yeast strains are selected for traits that enhance their application in quality production processes. Wine yeasts are required to survive in the harsh environment of fermenting grape must, while at the same time contributing to wine quality by producing desirable aromas and flavors. For this reason, there are hundreds of wine yeasts available, exhibiting characteristics that make them suitable for different fermentation conditions and winemaking practices. As wine styles evolve and technical winemaking requirements change, however, it becomes necessary to improve existing strains. This becomes a laborious and costly process when the targets for improvement involve flavor compound production. Here, we demonstrate a new approach harnessing preexisting industrial yeast strains that carry desirable flavor phenotypes - low hydrogen sulfide (H(2) S) production and high ester production. A low-H(2) S Saccharomyces cerevisiae strain previously generated by chemical mutagenesis was hybridized independently with two ester-producing natural interspecies hybrids of S. cerevisiae and Saccharomyces kudriavzevii. Deficiencies in sporulation frequency and spore viability were overcome through use of complementary selectable traits, allowing successful isolation of several novel hybrids exhibiting both desired traits in a single round of selection. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. Isolation and characterization of ethanol tolerant yeast strains

    PubMed Central

    Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

    2013-01-01

    Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092

  13. Comparison of DNA-based techniques for differentiation of production strains of ale and lager brewing yeast.

    PubMed

    Kopecká, J; Němec, M; Matoulková, D

    2016-06-01

    Brewing yeasts are classified into two species-Saccharomyces pastorianus and Saccharomyces cerevisiae. Most of the brewing yeast strains are natural interspecies hybrids typically polyploids and their identification is thus often difficult giving heterogenous results according to the method used. We performed genetic characterization of a set of the brewing yeast strains coming from several yeast culture collections by combination of various DNA-based techniques. The aim of this study was to select a method for species-specific identification of yeast and discrimination of yeast strains according to their technological classification. A group of 40 yeast strains were characterized using PCR-RFLP analysis of ITS-5·8S, NTS, HIS4 and COX2 genes, multiplex PCR, RAPD-PCR of genomic DNA, mtDNA-RFLP and electrophoretic karyotyping. Reliable differentiation of yeast to the species level was achieved by PCR-RFLP of HIS4 gene. Numerical analysis of the obtained RAPD-fingerprints and karyotype revealed species-specific clustering corresponding with the technological classification of the strains. Taxonomic position and partial hybrid nature of strains were verified by multiplex PCR. Differentiation among species using the PCR-RFLP of ITS-5·8S and NTS region was shown to be unreliable. Karyotyping and RFLP of mitochondrial DNA evinced small inaccuracies in strain categorization. PCR-RFLP of HIS4 gene and RAPD-PCR of genomic DNA are reliable and suitable methods for fast identification of yeast strains. RAPD-PCR with primer 21 is a fast and reliable method applicable for differentiation of brewing yeasts with only 35% similarity of fingerprint profile between the two main technological groups (ale and lager) of brewing strains. It was proved that PCR-RFLP method of HIS4 gene enables precise discrimination among three technologically important Saccharomyces species. Differentiation of brewing yeast to the strain level can be achieved using the RAPD-PCR technique. © 2016 The

  14. Estrogenic activities of chemicals related to food contact plastics and rubbers tested by the yeast two-hybrid assay.

    PubMed

    Ogawa, Yuko; Kawamura, Yoko; Wakui, Chiseko; Mutsuga, Motoh; Nishimura, Tetsuji; Tanamoto, Kenichi

    2006-04-01

    Food contact plastics and rubbers possibly contain many kinds of chemicals such as monomers, oligomers, additives, degradation products of polymers and additives, and impurities. Among them, bisphenol A, nonylphenol, benzylbutyl phthalate, styrene oligomers and hydroxylated benzophenones have been reported to possess estrogenic activities. In this study, other chemicals related to food contact plastics and rubbers, and their metabolites induced by the S9-mixture were tested for their estrogenic activities using the yeast two-hybrid assay. Among the 150 chemicals, 10 chemicals such as bis(4-hydroxyphenyl) methane, 4-cyclohexylphenol, 4-phenylphenol, 4,4'-isopropylidenediphenol alkylphosphite, two type of styrenated phenol (including mono type), tris(nonylphenyl) phosphite, 2,2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone and 2,4-diphenyl-4-methyl-1-pentene, their metabolites and the metabolites of 6 other chemicals, such as 2-(phenylmethyl) phenol, styrenated phenol (di and tri type), 1-(N-phenylamino)naphthalene, 4-tert-butylphenylsalicylate, nonylphenol ethoxylates and 2-methyl-6-tert-butylphenol, displayed estrogenic activities. All of them contained a phenol group in their chemical structures or formed one easily by hydrolysis or metabolism. However, most of the chemicals related to food contact plastics and rubbers, and their metabolites did not show any estrogenicity.

  15. From drug to protein: using yeast genetics for high-throughput target discovery.

    PubMed

    Armour, Christopher D; Lum, Pek Yee

    2005-02-01

    The budding yeast Saccharomyces cerevisiae has long been an effective eukaryotic model system for understanding basic cellular processes. The genetic tractability and ease of manipulation in the laboratory make yeast well suited for large-scale chemical and genetic screens. Several recent studies describing the use of yeast genetics for high-throughput drug target identification are discussed in this review.

  16. Degradation of lindane by a novel embedded bio-nano hybrid system in aqueous environment.

    PubMed

    Salam, Jaseetha Abdul; Das, Nilanjana

    2015-03-01

    The objective of this study was to evaluate the effect of an embedded bio-nano hybrid system using nanoscale zinc oxide (n-ZnO) and lindane-degrading yeast Candida VITJzN04 for lindane degradation. Nano-embedding of the yeast was done with chemically synthesized n-ZnO particles (50 mg/mL) and was visualized by atomic force microscope (AFM) and scanning electron microscope (SEM). Nanoparticles were embedded substantially on the surfaces of the yeast cells and translocated into the cell cytoplasm without causing any lethal effect to the cell until 50 mg/mL. Lindane (600 mg/L) degradation was studied both in the individual and hybrid system. Rapid reductive-dechlorination of lindane was attained with n-ZnO under illuminated conditions, with the generation of chlorobenzene and benzene as dechlorination products. The bio-nano hybrid was found to be more effective compared to the native yeasts for lindane degradation and resulted in complete removal within 3 days. The kinetic data analysis implied that the half-life of lindane was 9 h for bio-nano hybrid and 28 h for Candida VITJzN04. The enhanced lindane degradation by bio-nano hybrid might be due to increased porosity and permeability of the yeast cell membrane, facilitating the easy entry of lindane into cell cytoplasm and n-ZnO-mediated dechlorination. To the best of our knowledge, this report, for the first time, suggests the use of n-ZnO-mediated dechlorination of lindane and the novel bio-nano hybrid system that reduces the half-life to one third of the time taken by the yeast alone. The embedded bio-nano hybrid system may be exploited as an effective remediation tool for the treatment of lindane-contaminated wastewaters.

  17. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    PubMed

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  18. Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

    PubMed

    Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Altelaar, A F Maarten; van Leeuwen, Fred

    2016-12-06

    Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

  19. Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

    PubMed Central

    Ohnuki, Shinsuke; Okada, Hiroki; Friedrich, Anne; Kanno, Yoichiro; Goshima, Tetsuya; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Tamura, Hiroyasu; Nakamura, Ryo; Hirata, Dai; Fukuda, Hisashi; Shimoi, Hitoshi; Kitamoto, Katsuhiko; Watanabe, Daisuke; Schacherer, Joseph; Akao, Takeshi; Ohya, Yoshikazu

    2017-01-01

    Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies. PMID:28642365

  20. Structural and electronic properties of U{sub n}O{sub m} (n=1-3,m=1-3n) clusters: A theoretical study using screened hybrid density functional theory

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Yu; Liu, Haitao; Zhang, Ping, E-mail: zhang-ping@iapcm.ac.cn

    The structural and electronic properties of small uranium oxide clusters U{sub n}O{sub m} (n=1-3, m=1-3n) are systematically studied within the screened hybrid density functional theory. It is found that the formation of U–O–U bondings and isolated U–O bonds are energetically more stable than U–U bondings. As a result, no uranium cores are observed. Through fragmentation studies, we find that the U{sub n}O{sub m} clusters with the m/n ratio between 2 and 2.5 are very stable, hinting that UO{sub 2+x} hyperoxides are energetically stable. Electronically, we find that the O-2p states always distribute in the deep energy range, and the U-5fmore » states always distribute at the two sides of the Fermi level. The U-6d states mainly hybridize with the U-5f states in U-rich clusters, while hybridizing with O-2p states in O-rich clusters. Our work is the first one on the screened hybrid density functional theory level studying the atomic and electronic properties of the actinide oxide clusters.« less

  1. Variation in α-acetolactate production within the hybrid lager yeast group Saccharomyces pastorianus and affirmation of the central role of the ILV6 gene.

    PubMed

    Gibson, Brian; Krogerus, Kristoffer; Ekberg, Jukka; Monroux, Adrien; Mattinen, Laura; Rautio, Jari; Vidgren, Virve

    2015-01-01

    A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Screening in crystalline liquids protects energetic carriers in hybrid perovskites

    NASA Astrophysics Data System (ADS)

    Zhu, Haiming; Miyata, Kiyoshi; Fu, Yongping; Wang, Jue; Joshi, Prakriti; Niesner, Daniel; Williams, Kristopher; Jin, Song; Zhu, Xiaoyang

    Hybrid lead halide perovskites exhibit carrier properties that resemble those of pristine nonpolar semiconductors despite static and dynamic disorder, but how carriers are protected from efficient scattering with charged defects and optical phonons is unknown. Here, we reveal the carrier protection mechanism by comparing three single-crystal lead bromide perovskites: CH3NH3PbBr3, CH(NH2)2PbBr3, and CsPbBr3. We observed hot fluorescence emission from energetic carriers with 102 picosecond lifetimes in CH3NH3PbBr3 or CH(NH,SUB>2)2PbBr3, but not in CsPbBr3. The hot fluorescence is correlated with liquid-like molecular reorientational motions, suggesting that dynamic screening protects energetic carriers via solvation or large polaron formation on time scales competitive with that of ultrafast cooling. Similar protections likely exist for band-edge carriers. The long-lived energetic carriers may enable hot-carrier solar cells with efficiencies exceeding the Shockley-Queisser limit. This work was supported by U.S. Department of Energy Grant ER46980, National Science Foundation, Grant DMR 1420634 (MRSEC), and Department of Energy Award DE-FG02-09ER46664.

  3. The dynamic three-dimensional organization of the diploid yeast genome

    PubMed Central

    Kim, Seungsoo; Liachko, Ivan; Brickner, Donna G; Cook, Kate; Noble, William S; Brickner, Jason H; Shendure, Jay; Dunham, Maitreya J

    2017-01-01

    The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization. DOI: http://dx.doi.org/10.7554/eLife.23623.001 PMID:28537556

  4. Investigating flavour characteristics of British ale yeasts: techniques, resources and opportunities for innovation

    PubMed Central

    Parker, Neva; James, Steve; Dicks, Jo; Bond, Chris; Nueno-Palop, Carmen; White, Chris; Roberts, Ian N

    2015-01-01

    Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour-related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25361168

  5. Micropatterned 2D Hybrid Perovskite Thin Films with Enhanced Photoluminescence Lifetimes

    PubMed Central

    2018-01-01

    The application of luminescent materials in display screens and devices requires micropatterned structures. In this work, we have successfully printed microstructures of a two-dimensional (2D), orange-colored organic/inorganic hybrid perovskite ((C6H5CH2NH3)2PbI4) using two different soft lithography techniques. Notably, both techniques yield microstructures with very high aspect ratios in the range of 1.5–1.8. X-ray diffraction reveals a strong preferential orientation of the crystallites along the c-axis in both patterned structures, when compared to nonpatterned, drop-casted thin films. Furthermore, (time-resolved) photoluminescence (PL) measurements reveal that the optical properties of (C6H5CH2NH3)2PbI4 are conserved upon patterning. We find that the larger grain sizes of the patterned films with respect to the nonpatterned film give rise to an enhanced PL lifetime. Thus, our results demonstrate easy and cost-effective ways to manufacture patterns of 2D organic/inorganic hybrid perovskites, while even improving their optical properties. This demonstrates the potential use of color-tunable 2D hybrids in optoelectronic devices. PMID:29578335

  6. Micropatterned 2D Hybrid Perovskite Thin Films with Enhanced Photoluminescence Lifetimes.

    PubMed

    Kamminga, Machteld E; Fang, Hong-Hua; Loi, Maria Antonietta; Ten Brink, Gert H; Blake, Graeme R; Palstra, Thomas T M; Ten Elshof, Johan E

    2018-04-18

    The application of luminescent materials in display screens and devices requires micropatterned structures. In this work, we have successfully printed microstructures of a two-dimensional (2D), orange-colored organic/inorganic hybrid perovskite ((C 6 H 5 CH 2 NH 3 ) 2 PbI 4 ) using two different soft lithography techniques. Notably, both techniques yield microstructures with very high aspect ratios in the range of 1.5-1.8. X-ray diffraction reveals a strong preferential orientation of the crystallites along the c-axis in both patterned structures, when compared to nonpatterned, drop-casted thin films. Furthermore, (time-resolved) photoluminescence (PL) measurements reveal that the optical properties of (C 6 H 5 CH 2 NH 3 ) 2 PbI 4 are conserved upon patterning. We find that the larger grain sizes of the patterned films with respect to the nonpatterned film give rise to an enhanced PL lifetime. Thus, our results demonstrate easy and cost-effective ways to manufacture patterns of 2D organic/inorganic hybrid perovskites, while even improving their optical properties. This demonstrates the potential use of color-tunable 2D hybrids in optoelectronic devices.

  7. Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage.

    PubMed

    D'hooge, Petra; Coun, Catherina; Van Eyck, Vincent; Faes, Liesbeth; Ghillebert, Ruben; Mariën, Lore; Winderickx, Joris; Callewaert, Geert

    2015-08-01

    Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2014-10-01

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

  9. No evidence for extrinsic post-zygotic isolation in a wild Saccharomyces yeast system.

    PubMed

    Charron, Guillaume; Landry, Christian R

    2017-06-01

    Although microorganisms account for the largest fraction of Earth's biodiversity, we know little about how their reproductive barriers evolve. Sexual microorganisms such as Saccharomyces yeasts rapidly develop strong intrinsic post-zygotic isolation, but the role of extrinsic isolation in the early speciation process remains to be investigated. We measured the growth of F 1 hybrids between two incipient species of Saccharomyces paradoxus to assess the presence of extrinsic post-zygotic isolation across 32 environments. More than 80% of hybrids showed either partial dominance of the best parent or over-dominance for growth, revealing no fitness defects in F 1 hybrids. Extrinsic reproductive isolation therefore likely plays little role in limiting gene flow between incipient yeast species and is not a requirement for speciation. © 2017 The Author(s).

  10. Characterization of butter spoiling yeasts and their inhibition by some spices.

    PubMed

    Sagdic, Osman; Ozturk, Ismet; Bayram, Okan; Kesmen, Zulal; Yilmaz, Mustafa Tahsin

    2010-01-01

    This study was designed to identify the yeasts in packaged and unpackaged butters and screen antiyeast activity of spices, including marjoram (Origanum majorana L.), summer savory (Satureja hortensis L.), and black cumin (Nigella sativa L.) against the most dominant yeast species in the packaged and unpackaged butters. Mean total yeast populations were 5.40 log CFU/g in unpackaged butter samples and 2.22 log CFU/g in packaged butter samples, indicating better hygienic quality of packaged samples. Forty-nine yeast species were isolated and identified from butter samples with the most prevalent isolates belonging to genera Candida-C. kefyr, C. zeylanoides, and C. lambica-and with moderate number of isolates belonging to genera Cryptococcus, Rhodotorula, Saccharomyces, and Zygosaccharomyces. Black cumin exhibited the highest antiyeast activity against C. zeylanoides and C. lambica species, even inhibited these species, while summer savory inhibited C. kefyr. The results of this study revealed clear antimicrobial potential of black cumin against the yeast species isolated from butters. Marjoram, summer savory, and black cumin could be used as natural antimicrobial agents against spoilage yeasts in food preservation, especially in butter. © 2010 Institute of Food Technologists®

  11. An original method for producing acetaldehyde and diacetyl by yeast fermentation.

    PubMed

    Rosca, Irina; Petrovici, Anca Roxana; Brebu, Mihai; Stoica, Irina; Minea, Bogdan; Marangoci, Narcisa

    In this study a natural culture medium that mimics the synthetic yeast peptone glucose medium used for yeast fermentations was designed to screen and select yeasts capable of producing high levels of diacetyl and acetaldehyde. The presence of whey powder and sodium citrate in the medium along with manganese and magnesium sulfate enhanced both biomass and aroma development. A total of 52 yeasts strains were cultivated in two different culture media, namely, yeast peptone glucose medium and yeast acetaldehyde-diacetyl medium. The initial screening of the strains was based on the qualitative reaction of the acetaldehyde with Schiff's reagent (violet color) and diacetyl with Brady's reagent (yellow precipitate). The fermented culture media of 10 yeast strains were subsequently analyzed by gas chromatography to quantify the concentration of acetaldehyde and diacetyl synthesized. Total titratable acidity values indicated that a total titratable acidity of 5.5°SH, implying culture medium at basic pH, was more favorable for the acetaldehyde biosynthesis using strain D15 (Candida lipolytica; 96.05mgL -1 acetaldehyde) while a total titratable acidity value of 7°SH facilitated diacetyl flavor synthesis by strain D38 (Candida globosa; 3.58mgL -1 diacetyl). Importantly, the results presented here suggest that this can be potentially used in the baking industry. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  12. Formation by yeast of 2-furanmethanethiol and ethyl 2-mercaptopropionate aroma compounds in Japanese soy sauce.

    PubMed

    Meng, Qi; Hatakeyama, Makoto; Sugawara, Etsuko

    2014-01-01

    Two aroma compounds of volatile thiols, 2-furanmethanethiol (2FM) and ethyl 2-mercaptopropionate (ET2MP), were formed in five types of Japanese soy sauce during fermentation by yeast. The concentrations of 2FM and ET2MP in the soy sauce samples increased during alcoholic fermentation. The concentrations of 2FM and ET2MP were higher in the soy sauce fermented by Zygosaccharomyces rouxii than in that fermented by Candida versatilis. The enantiomers of ET2MP were separated by gas chromatography in a capillary column. The average enantiomeric ratio of ET2MP in the soy sauce was approximately 1:1. 2FM was formed by yeast in a medium prepared from cysteine and furfural, and cysteine is considered the key precursor of 2FM by yeast in soy sauce.

  13. Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast*

    PubMed Central

    Burr, Risa; Stewart, Emerson V.; Shao, Wei; Zhao, Shan; Hannibal-Bach, Hans Kristian; Ejsing, Christer S.; Espenshade, Peter J.

    2016-01-01

    Eukaryotic lipid synthesis is oxygen-dependent with cholesterol synthesis requiring 11 oxygen molecules and fatty acid desaturation requiring 1 oxygen molecule per double bond. Accordingly, organisms evaluate oxygen availability to control lipid homeostasis. The sterol regulatory element-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis in response to changing sterol and oxygen levels. However, notably missing is an SREBP-1 analog that regulates triacylglycerol and glycerophospholipid homeostasis in response to low oxygen. Consistent with this, studies have shown that the Sre1 transcription factor regulates only a fraction of all genes up-regulated under low oxygen. To identify new regulators of low oxygen adaptation, we screened the S. pombe nonessential haploid deletion collection and identified 27 gene deletions sensitive to both low oxygen and cobalt chloride, a hypoxia mimetic. One of these genes, mga2, is a putative transcriptional activator. In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid saturation. Indeed, addition of exogenous oleic acid to mga2Δ cells rescued the observed growth defects. Together, these results establish Mga2 as a transcriptional regulator of triacylglycerol and glycerophospholipid homeostasis in S. pombe, analogous to mammalian SREBP-1. PMID:27053105

  14. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

    2016-01-01

    HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

  15. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

  16. A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

    PubMed

    Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

    2016-01-01

    Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

  17. Modulation of molecular hybridization and charge screening in a carbon nanotube network channel using the electrical pulse method.

    PubMed

    Woo, Jun-Myung; Kim, Seok Hyang; Chun, Honnggu; Kim, Sung Jae; Ahn, Jinhong; Park, Young June

    2013-09-21

    In this paper, we investigate the effect of electrical pulse bias on DNA hybridization events in a biosensor platform, using a Carbon Nanotube Network (CNN) and Gold Nano Particles (GNP) as an electrical channel. The scheme provides both hybridization rate enhancement of bio molecules, and electrical measurement in a transient state to avoid the charge screening effect, thereby significantly improving the sensitivity. As an example, the probe DNA molecules oscillate with pulse trains, resulting in the enhancement of DNA hybridization efficiency, and accordingly of the sensor performances in Tris-EDTA (TE) buffer solution, by as much as over three times, compared to the non-biasing conditions. More importantly, a wide dynamic range of 10(6) (target-DNA concentration from 5 pM to 5 μM) is achieved in human serum. In addition, the pulse biasing method enables one to obtain the conductance change, before the ions within the Electrical Double Layer (EDL) are redistributed, to avoid the charge screening effect, leading to an additional sensitivity enhancement.

  18. Yeast Genomics for Bread, Beer, Biology, Bucks and Breath

    NASA Astrophysics Data System (ADS)

    Sakharkar, Kishore R.; Sakharkar, Meena K.

    The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

  19. The Yeast Deletion Collection: A Decade of Functional Genomics

    PubMed Central

    Giaever, Guri; Nislow, Corey

    2014-01-01

    The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. PMID:24939991

  20. MdHIR proteins repress anthocyanin accumulation by interacting with the MdJAZ2 protein to inhibit its degradation in apples

    PubMed Central

    Chen, Ke-Qin; Zhao, Xian-Yan; An, Xiu-Hong; Tian, Yi; Liu, Dan-Dan; You, Chun-Xiang; Hao, Yu-Jin

    2017-01-01

    In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis. PMID:28317851

  1. Yeast as a model system for mammalian seven-transmembrane segment receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jeansonne, N.E.

    1994-05-01

    Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomalmore » location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.« less

  2. LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

    PubMed Central

    Gu, Mingyu; LaJoie, Dollie; Chen, Opal S.; von Appen, Alexander; Ladinsky, Mark S.; Redd, Michael J.; Nikolova, Linda; Bjorkman, Pamela J.; Sundquist, Wesley I.; Ullman, Katharine S.; Frost, Adam

    2017-01-01

    Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope. PMID:28242692

  3. Sphingolipid biosynthesis upregulation by TOR Complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress

    PubMed Central

    Guerreiro, Joana F.; Muir, Alexander; Ramachandran, Subramaniam; Thorner, Jeremy; Sá-Correia, Isabel

    2016-01-01

    Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus, understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for S. cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the Target of Rapamycin (TOR) Complex 2 (TORC2). We show here by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of L-serine: palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus, appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks. PMID:27671892

  4. Diversity in Genetic In Vivo Methods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

    PubMed Central

    Stynen, Bram; Tournu, Hélène; Tavernier, Jan

    2012-01-01

    Summary: The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays. PMID:22688816

  5. Discovery of synthesis and secretion of polyol esters of fatty acids by four basidiomycetous yeast species in the order Sporidiobolales.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Fiehn, Oliver; Cathcart, Erin; Fry, Russell W; Kanti, Atit; Joko Nugroho, Agustinus; Faulina, Sarah Asih; Stephanandra, Sira; German, J Bruce; Boundy-Mills, Kyria L

    2017-06-01

    Polyol esters of fatty acids (PEFA) are amphiphilic glycolipids produced by yeast that could play a role as natural, environmentally friendly biosurfactants. We recently reported discovery of a new PEFA-secreting yeast species, Rhodotorula babjevae, a basidiomycetous yeast to display this behavior, in addition to a few other Rhodotorula yeasts reported on the 1960s. Additional yeast species within the taxonomic order Sporidiobolales were screened for secreted glycolipid production. PEFA production equal or above 1 g L -1 were detected in 19 out of 65 strains of yeast screened, belonging to 6 out of 30 yeast species tested. Four of these species were not previously known to secrete glycolipids. These results significantly increase the number of yeast species known to secrete PEFA, holding promise for expanding knowledge of PEFA synthesis and secretion mechanisms, as well as setting the groundwork towards commercialization.

  6. Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

    PubMed Central

    Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Maarten Altelaar, AF; van Leeuwen, Fred

    2016-01-01

    Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001 PMID:27922451

  7. Yeast as a potential vehicle for neglected tropical disease drug discovery.

    PubMed

    Denny, P W; Steel, P G

    2015-01-01

    High-throughput screening (HTS) efforts for neglected tropical disease (NTD) drug discovery have recently received increased attention because several initiatives have begun to attempt to reduce the deficit in new and clinically acceptable therapies for this spectrum of infectious diseases. HTS primarily uses two basic approaches, cell-based and in vitro target-directed screening. Both of these approaches have problems; for example, cell-based screening does not reveal the target or targets that are hit, whereas in vitro methodologies lack a cellular context. Furthermore, both can be technically challenging, expensive, and difficult to miniaturize for ultra-HTS [(u)HTS]. The application of yeast-based systems may overcome some of these problems and offer a cost-effective platform for target-directed screening within a eukaryotic cell context. Here, we review the advantages and limitations of the technologies that may be used in yeast cell-based, target-directed screening protocols, and we discuss how these are beginning to be used in NTD drug discovery. © 2014 Society for Laboratory Automation and Screening.

  8. A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression.

    PubMed

    Sugiyama, Yuka; Ikeshita, Nobuko; Shibahara, Hiromi; Yamamoto, Daisuke; Kawagishi, Mayuko; Iguchi, Genzo; Iida, Keiji; Takahashi, Yutaka; Kaji, Hidesuke; Chihara, Kazuo; Okimura, Yasuhiko

    2013-08-25

    PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  9. Sponge Sampling with Fluorescent In Situ Hybridization as a Screening Tool for the Early Detection of Esophageal Cancer.

    PubMed

    Haisley, Kelly R; Dolan, James P; Olson, Susan B; Toledo-Valdovinos, Sergio A; Hart, Kyle D; Bakis, Gene; Enestvedt, Brintha K; Hunter, John G

    2017-02-01

    Sponge cytology is a novel screening tool for esophageal cancer but has been unable to be validated for widespread use. Our aim was to apply fluorescent in situ hybridization to sponge cytology samples in order to evaluate the safety and efficacy of this modality in screening for esophageal cancer. At a single, multidisciplinary, NCI-designated cancer center, patients completed sponge cytology sampling prior to upper endoscopy. Samples were analyzed by p53 fluorescent in situ hybridization, and results were compared to the endoscopic diagnosis. Fifty patients were enrolled (96 % Caucasian, 68 % male, median age of 67). All patients successfully swallowed the capsule. No complications (string breakage, bleeding, mucosal injury) occurred. Endoscopy revealed that 38 % had normal esophageal mucosa and 62 % had an esophageal mucosal abnormality. In total, six samples demonstrated p53 loss (94 % specificity for any abnormality). The sensitivity of the p53 fluorescent in situ hybridization probe was13.3 % for any abnormality, 10 % for intestinal metaplasia, and 0 % for dysplasia or esophageal cancer. Esophageal sponge cytology is a promising, safe, and tolerable method for collecting esophageal cell samples. However, our data suggest that p53 fluorescent in situ hybridization does not improve the sensitivity for detecting cancer in these samples.

  10. Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

    PubMed

    Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

    2015-10-01

    Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

  11. Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

    PubMed Central

    Li, Liang; Mustafi, Debarshi; Fu, Qiang; Tereshko, Valentina; Chen, Delai L.; Tice, Joshua D.; Ismagilov, Rustem F.

    2006-01-01

    High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a “hybrid” droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as ≈140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in ≈10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, ≈1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization. PMID:17159147

  12. Mitochondrial Recombination and Introgression during Speciation by Hybridization.

    PubMed

    Leducq, Jean-Baptiste; Henault, Mathieu; Charron, Guillaume; Nielly-Thibault, Lou; Terrat, Yves; Fiumera, Heather L; Shapiro, B Jesse; Landry, Christian R

    2017-08-01

    Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. 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

  14. Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)*

    PubMed Central

    Jiang, Ya-Jun; Che, Mei-Xia; Yuan, Jin-Qiao; Xie, Yuan-Yuan; Yan, Xian-Zhong; Hu, Hong-Yu

    2011-01-01

    Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD. PMID:21566141

  15. Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA).

    PubMed

    Jiang, Ya-Jun; Che, Mei-Xia; Yuan, Jin-Qiao; Xie, Yuan-Yuan; Yan, Xian-Zhong; Hu, Hong-Yu

    2011-07-15

    Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.

  16. Yeast as a model to study apoptosis?

    PubMed

    Fleury, Christophe; Pampin, Mathieu; Tarze, Agathe; Mignotte, Bernard

    2002-02-01

    Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms. The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, ...) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis. Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics). This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.

  17. Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins.

    PubMed

    Hoggard, Timothy A; Chang, FuJung; Perry, Kelsey Rae; Subramanian, Sandya; Kenworthy, Jessica; Chueng, Julie; Shor, Erika; Hyland, Edel M; Boeke, Jef D; Weinreich, Michael; Fox, Catherine A

    2018-05-01

    Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

  18. Genetic relationship and biological status of the industrially important yeast Saccharomyces eubayanus Sampaio et al.

    PubMed

    Naumov, G I

    2017-03-01

    The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.

  19. Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

    PubMed

    Maier, Richard H; Maier, Christina J; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2012-12-01

    Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Gene-nutrient interaction markedly influences yeast chronological lifespan.

    PubMed

    Smith, Daniel L; Maharrey, Crystal H; Carey, Christopher R; White, Richard A; Hartman, John L

    2016-12-15

    Research into the genetic mechanisms of aging has expanded rapidly over the past two decades. This has in part been the result of the use of model organisms (particularly yeast, worms and flies) and high-throughput technologies, combined with a growing interest in aging research. Despite this progress, widespread consensus regarding the pathways that are fundamental to the modulation of cellular aging and lifespan for all organisms has been limited due to discrepancies between different studies. We have compared results from published genome-wide, chronological lifespan (CLS) screens of individual gene deletion strains in Saccharomyces cerevisiae in order to identify gene deletion strains with consistent influences on longevity as possible indicators of fundamental aging processes from this single-celled, eukaryotic model organism. Three previous reports have described genetic modifiers of chronological aging in the budding yeast (S. cerevisiae) using the yeast gene deletion strain collection. We performed a comparison among the data sets using correlation and decile distribution analysis to describe concordance between screens and identify strains that consistently increased or decreased CLS. We used gene enrichment analysis in an effort to understand the biology underlying genes identified in multiple studies. We attempted to replicate the different experimental conditions employed by the screens to identify potential sources of variability in CLS worth further investigating. Among 3209 strains present in all three screens, nine deletions strains were in common in the longest-lived decile (2.80%) and thirteen were in common in the shortest-lived decile (4.05%) of all three screens. Similarly, pairwise overlap between screens was low. When the same comparison was extended to three deciles to include more mutants studied in common between the three screens, enrichment of cellular processes based on gene ontology analysis in the long-lived strains remained very

  1. Gene-Nutrient Interaction Markedly Influences Yeast Chronological Lifespan

    PubMed Central

    Smith, Daniel L.; Maharrey, Crystal H.; Carey, Christopher R.; White, Richard A.; Hartman, John L.

    2016-01-01

    Purpose Research into the genetic mechanisms of aging has expanded rapidly over the past two decades. This has in part been the result of the use of model organisms (particularly yeast, worms and flies) and high-throughput technologies, combined with a growing interest in aging research. Despite this progress, widespread consensus regarding the pathways that are fundamental to the modulation of cellular aging and lifespan for all organisms has been limited due to discrepancies between different studies. We have compared results from published genome-wide, chronological lifespan (CLS) screens of individual gene deletion strains in S. cerevisiae in order to identify gene deletion strains with consistent influences on longevity as possible indicators of fundamental aging processes from this single-celled, eukaryotic model organism. Methods Three previous reports have described genetic modifiers of chronological aging in the budding yeast (S. cerevisiae) using the yeast gene deletion strain collection. We performed a comparison among the data sets using correlation and decile distribution analysis to describe concordance between screens and identify strains that consistently increased or decreased CLS. We used gene enrichment analysis in an effort to understand the biology underlying genes identified in multiple studies. We attempted to replicate the different experimental conditions employed by the screens to identify potential sources of variability in CLS worth further investigating. Results Among 3209 strains present in all three screens, nine (2.80%) deletions strains were in common in the longest-lived decile and thirteen (4.05%) were in common in the shortest-lived decile for all three screens. Similarly, pairwise overlap between screens was low. When the same comparison was extended to three deciles to include more mutants studied in common between the three screens, enrichment of cellular processes based on gene ontology analysis in the long-lived strains

  2. [Novel hybrid inhibitors of the phage T7 RNA polymerase: synthesis, docking and screening in vitro].

    PubMed

    Kostina, V H; Pal'chykovs'ka, L H; Platonov, M O; Vasyl'chenko, O V; Lysenko, N A; Alekseeva, I V

    2012-01-01

    A number of new hybrid heteroaromatic compounds, consisting of tricyclic fragments (acridone, thioxanthone and phenazine) and bicyclic fragments (benzimidazole, benzothiazole and benzoxazole) were synthesized using the method, developed by the authors. As a result of screening against the transcription model system of the phage T7 DNA-dependent RNA polymerase three effective inhibitors of the RNA syntheses with the IC50 value of 8.9, 5.7 and 19.8 microM were detected. To cast light on the mode of interaction between the synthesized compounds and the target, the molecular docking was applied to the model pocket of the phage T7 RNA polymerase transcription complex. It was established that these ligands form networks of H-bonds with residues of the pocket conservative amino acids and pi-interaction with the Mg2+ ion. A planar geometry of the hybrid molecules, realized due to the intramolecular H-bonds, proved to be an important structural feature, which correlates with an efficacious inhibitory activity.

  3. The Yeast Eukaryotic Translation Initiation Factor 2B Translation Initiation Complex Interacts with the Fatty Acid Synthesis Enzyme YBR159W and Endoplasmic Reticulum Membranes

    PubMed Central

    Browne, Christopher M.; Samir, Parimal; Fites, J. Scott; Villarreal, Seth A.

    2013-01-01

    Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm. PMID:23263984

  4. Affordable proteomics: the two-hybrid systems.

    PubMed

    Gillespie, Marc

    2003-06-01

    Numerous proteomic methodologies exist, but most require a heavy investment in expertise and technology. This puts these approaches out of reach for many laboratories and small companies, rarely allowing proteomics to be used as a pilot approach for biomarker or target identification. Two proteomic approaches, 2D gel electrophoresis and the two-hybrid systems, are currently available to most researchers. The two-hybrid systems, though accommodating to large-scale experiments, were originally designed as practical screens, that by comparison to current proteomics tools were small-scale, affordable and technically feasible. The screens rapidly generated data, identifying protein interactions that were previously uncharacterized. The foundation for a two-hybrid proteomic investigation can be purchased as separate kits from a number of companies. The true power of the technique lies not in its affordability, but rather in its portability. The two-hybrid system puts proteomics back into laboratories where the output of the screens can be evaluated by researchers with experience in the particular fields of basic research, cancer biology, toxicology or drug development.

  5. Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE 3‐1

    PubMed Central

    Troisi, Elizabeth M.; Rockman, Michael E.; Nguyen, Phil P.; Oliver, Emily E.

    2015-01-01

    Summary Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion‐specific requirements for the propagation of the [URE 3] prion variant [URE 3‐1], we screened 12 yeast cytosolic J‐proteins, and here we report a novel role for the J‐protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE 3‐1] is specifically dependent upon Swa2, but not on any of the 11 other J‐proteins. Further, we show that [URE 3‐1] propagation requires both a functional J‐domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2‐clathrin binding. Because the J‐domain of Swa2 can be replaced with the J‐domains of other proteins, our data strongly suggest that prion‐chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation. PMID:26031938

  6. Lipase production in lipolytic yeast from Wonorejo mangrove area

    NASA Astrophysics Data System (ADS)

    Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya

    2017-06-01

    Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.

  7. Implication of Ca2+ in the regulation of replicative life span of budding yeast.

    PubMed

    Tsubakiyama, Ryohei; Mizunuma, Masaki; Gengyo, Anri; Yamamoto, Josuke; Kume, Kazunori; Miyakawa, Tokichi; Hirata, Dai

    2011-08-19

    In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.

  8. Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches’ Broom disease

    PubMed Central

    de Souza Cabral, Anderson; de Carvalho, Patricia Maria Barroso; Pinotti, Tatiana; Hagler, Allen Norton; Mendonça-Hagler, Leda Cristina Santana; Macrae, Andrew

    2009-01-01

    Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth. PMID:24031327

  9. Screened exchange hybrid density functional for accurate and efficient structures and interaction energies.

    PubMed

    Brandenburg, Jan Gerit; Caldeweyher, Eike; Grimme, Stefan

    2016-06-21

    We extend the recently introduced PBEh-3c global hybrid density functional [S. Grimme et al., J. Chem. Phys., 2015, 143, 054107] by a screened Fock exchange variant based on the Henderson-Janesko-Scuseria exchange hole model. While the excellent performance of the global hybrid is maintained for small covalently bound molecules, its performance for computed condensed phase mass densities is further improved. Most importantly, a speed up of 30 to 50% can be achieved and especially for small orbital energy gap cases, the method is numerically much more robust. The latter point is important for many applications, e.g., for metal-organic frameworks, organic semiconductors, or protein structures. This enables an accurate density functional based electronic structure calculation of a full DNA helix structure on a single core desktop computer which is presented as an example in addition to comprehensive benchmark results.

  10. The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

    PubMed

    Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

    2010-04-01

    We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

  11. A meta-GGA level screened range-separated hybrid functional by employing short range Hartree-Fock with a long range semilocal functional.

    PubMed

    Jana, Subrata; Samal, Prasanjit

    2018-03-28

    The range-separated hybrid density functionals are very successful in describing a wide range of molecular and solid-state properties accurately. In principle, such functionals are designed from spherically averaged or system averaged as well as reverse engineered exchange holes. In the present attempt, the screened range-separated hybrid functional scheme has been applied to the meta-GGA rung by using the density matrix expansion based semilocal exchange hole (or functional). The hybrid functional proposed here utilizes the spherically averaged density matrix expansion based exchange hole in the range separation scheme. For slowly varying density correction the range separation scheme is employed only through the local density approximation based exchange hole coupled with the corresponding fourth order gradient approximate Tao-Mo enhancement factor. The comprehensive testing and performance of the newly constructed functional indicates its applicability in describing several molecular properties. The most appealing feature of this present screened hybrid functional is that it will be practically very useful in describing solid-state properties at the meta-GGA level.

  12. Hybridization within Saccharomyces Genus Results in Homoeostasis and Phenotypic Novelty in Winemaking Conditions

    PubMed Central

    Dillmann, Christine; Bely, Marina; la Guerche, Stéphane; Giraud, Christophe; Huet, Sylvie; Sicard, Delphine; Masneuf-Pomarede, Isabelle; de Vienne, Dominique; Marullo, Philippe

    2015-01-01

    Despite its biotechnological interest, hybridization, which can result in hybrid vigor, has not commonly been studied or exploited in the yeast genus. From a diallel design including 55 intra- and interspecific hybrids between Saccharomyces cerevisiae and S. uvarum grown at two temperatures in enological conditions, we analyzed as many as 35 fermentation traits with original statistical and modeling tools. We first showed that, depending on the types of trait – kinetics parameters, life-history traits, enological parameters and aromas –, the sources of variation (strain, temperature and strain * temperature effects) differed in a large extent. Then we compared globally three groups of hybrids and their parents at two growth temperatures: intraspecific hybrids S. cerevisiae * S. cerevisiae, intraspecific hybrids S. uvarum * S. uvarum and interspecific hybrids S. cerevisiae * S. uvarum. We found that hybridization could generate multi-trait phenotypes with improved oenological performances and better homeostasis with respect to temperature. These results could explain why interspecific hybridization is so common in natural and domesticated yeast, and open the way to applications for wine-making. PMID:25946464

  13. Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.

    PubMed

    Méndez, S P; González, E B; Sanz-Medel, A

    2001-05-01

    Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast. Copyright 2001 John Wiley & Sons, Ltd.

  14. Brewer's Yeast Improves Glycemic Indices in Type 2 Diabetes Mellitus.

    PubMed

    Hosseinzadeh, Payam; Javanbakht, Mohammad Hassan; Mostafavi, Seyed-Ali; Djalali, Mahmoud; Derakhshanian, Hoda; Hajianfar, Hossein; Bahonar, Ahmad; Djazayery, Abolghassem

    2013-10-01

    Brewer's yeast may have beneficial effects on insulin receptors because of itsglucose tolerance factor in diabetic patients. This study was conducted to investigate the effects of brewer's yeast supplementation on glycemic indices in patients with type 2 diabetes mellitus. In a randomized double-blind controlled clinical trial, 84 adults (21 men and 63 women) aged 46.3 ± 6.1 years old with type 2 diabetes mellitus were recruited and divided randomly into two groups: Supplement group receiving brewer's yeast (six 300mg tablets/day, total 1800 mg) and control group receiving placebo (six 300mg tablets/day) for 12 weeks. Body weight, height, body mass index, food consumption (based on 24h food record), fasting blood sugar (FBS), glycosylated hemoglobin, insulin sensitivity, and insulin resistance were measured before and after the intervention. Data analysis was performed using the Statistical Package for Social Sciences (version 18.0). The changes in FBS, glycosylated hemoglobin, and insulin sensitivity were significantly different between the two groups during the study (respectively P < 0.001, P < 0.001, P = 0.02 independent sample t-test). There was a significant difference in FBS, glycosylated hemoglobin, and insulin sensitivity at the end of the study between the two groups after removing the effects of baseline values (respectively P = 0.002, P < 0.001, P = 0.02, analysis of covariance). Changes in body mass index, 24h food record, insulin resistance were not significant. Dietary supplementation with brewer›s yeast besides the usual treatment of diabetes can ameliorate blood glucose variables in type 2 diabetes mellitus.

  15. Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory course in molecular biology.

    PubMed

    Gammie, Alison E; Erdeniz, Naz

    2004-01-01

    This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

  16. Cell-free fetal DNA screening in the USA: a cost analysis of screening strategies.

    PubMed

    Evans, M I; Sonek, J D; Hallahan, T W; Krantz, D A

    2015-01-01

    To determine whether implementation of primary cell-free fetal DNA (cffDNA) screening would be cost-effective in the USA and to evaluate potential lower-cost alternatives. Three strategies to screen for trisomy 21 were evaluated using decision tree analysis: 1) a primary strategy in which cffDNA screening was offered to all patients, 2) a contingent strategy in which cffDNA screening was offered only to patients who were high risk on traditional first-trimester screening and 3) a hybrid strategy in which cffDNA screening was offered to all patients ≥ 35 years of age and only to patients < 35 years who were high risk after first-trimester screening. Four traditional screening protocols were evaluated, each assessing nuchal translucency (NT) and pregnancy-associated plasma protein-A (PAPP-A) along with either free or total beta-human chorionic gonadotropin (β-hCG), with or without nasal bone (NB) assessment. Utilizing a primary cffDNA screening strategy, the cost per patient was 1017 US$. With a traditional screening protocol using free β-hCG, PAPP-A and NT assessment as part of a hybrid screening strategy, a contingent strategy with a 1/300 cut-off and a contingent strategy with a 1/1000 cut-off, the cost per patient was 474, 430 and 409 US$, respectively. Findings were similar using the other traditional screening protocols. Marginal cost per viable case detected for the primary screening strategy as compared to the other strategies was 3-16 times greater than the cost of care for a missed case. Primary cffDNA screening is not currently a cost-effective strategy. The contingent strategy was the lowest-cost alternative, especially with a risk cut-off of 1/1000. The hybrid strategy, although less costly than primary cffDNA screening, was more costly than the contingent strategy. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.

  17. 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.

  18. Antioxidant properties and global metabolite screening of the probiotic yeast Saccharomyces cerevisiae var. boulardii.

    PubMed

    Datta, Suprama; Timson, David J; Annapure, Uday S

    2017-07-01

    Saccharomyces cerevisiae var. boulardii is the only yeast species with probiotic properties. It is considered to have therapeutic significance in gastrointestinal disorders. In the present study, a comparative physiological study between this yeast and Saccharomyces cerevisiae (BY4742) was performed by evaluating two prominent traits of probiotic species, responses to different stress conditions and antioxidant capacity. A global metabolite profile was also developed aiming to identify which therapeutically important secondary metabolites are produced. Saccharomyces cerevisiae var. boulardii showed no significant difference in growth patterns but greater stress tolerance compared to S. cerevisiae. It also demonstrated a six- to 10-fold greater antioxidant potential (judged by the 1,1-diphenyl-2-picrylhydrazyl assay), with a 70-fold higher total phenolic content and a 20-fold higher total flavonoid content in the extracellular fraction. These features were clearly differentiated by principal component analysis and further indicated by metabolite profiling. The extracellular fraction of the S. cerevisiae var. boulardii cultures was found to be rich in polyphenolic metabolites: vanillic acid, cinnamic acid, phenyl ethyl alcohol (rose oil), erythromycin, amphetamine and vitamin B 6 , which results in the antioxidant capacity of this strain. The present study presents a new perspective for differentiating the two genetically related strains of yeast, S. cerevisiae and S. cerevisiae var. boulardii by assessing their metabolome fingerprints. In addition to the correlation of the phenotypic properties with the secretory metabolites of these two yeasts, the present study also emphasizes the potential to exploit S. cerevisiae var. boulardii in the industrial production of these metabolites. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. Novel Hybrid Virtual Screening Protocol Based on Molecular Docking and Structure-Based Pharmacophore for Discovery of Methionyl-tRNA Synthetase Inhibitors as Antibacterial Agents

    PubMed Central

    Liu, Chi; He, Gu; Jiang, Qinglin; Han, Bo; Peng, Cheng

    2013-01-01

    Methione tRNA synthetase (MetRS) is an essential enzyme involved in protein biosynthesis in all living organisms and is a potential antibacterial target. In the current study, the structure-based pharmacophore (SBP)-guided method has been suggested to generate a comprehensive pharmacophore of MetRS based on fourteen crystal structures of MetRS-inhibitor complexes. In this investigation, a hybrid protocol of a virtual screening method, comprised of pharmacophore model-based virtual screening (PBVS), rigid and flexible docking-based virtual screenings (DBVS), is used for retrieving new MetRS inhibitors from commercially available chemical databases. This hybrid virtual screening approach was then applied to screen the Specs (202,408 compounds) database, a structurally diverse chemical database. Fifteen hit compounds were selected from the final hits and shifted to experimental studies. These results may provide important information for further research of novel MetRS inhibitors as antibacterial agents. PMID:23839093

  20. Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

    PubMed

    Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

    2016-09-01

    Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

  1. Vapor-fed bio-hybrid fuel cell.

    PubMed

    Benyamin, Marcus S; Jahnke, Justin P; Mackie, David M

    2017-01-01

    Concentration and purification of ethanol and other biofuels from fermentations are energy-intensive processes, with amplified costs at smaller scales. To circumvent the need for these processes, and to potentially reduce transportation costs as well, we have previously investigated bio-hybrid fuel cells (FCs), in which a fermentation and FC are closely coupled. However, long-term operation requires strictly preventing the fermentation and FC from harming each other. We introduce here the concept of the vapor-fed bio-hybrid FC as a means of continuously extracting power from ongoing fermentations at ambient conditions. By bubbling a carrier gas (N 2 ) through a yeast fermentation and then through a direct ethanol FC, we protect the FC anode from the catalyst poisons in the fermentation (which are non-volatile), and also protect the yeast from harmful FC products (notably acetic acid) and from build-up of ethanol. Since vapor-fed direct ethanol FCs at ambient conditions have never been systematically characterized (in contrast to vapor-fed direct methanol FCs), we first assess the effects on output power and conversion efficiency of ethanol concentration, vapor flow rate, and FC voltage. The results fit a continuous stirred-tank reactor model. Over a wide range of ethanol partial pressures (2-8 mmHg), power densities are comparable to those for liquid-fed direct ethanol FCs at the same temperature, with power densities >2 mW/cm 2 obtained. We then demonstrate the continuous operation of a vapor-fed bio-hybrid FC with fermentation for 5 months, with no indication of performance degradation due to poisoning (of either the FC or the fermentation). It is further shown that the system is stable, recovering quickly from disturbances or from interruptions in maintenance. The vapor-fed bio-hybrid FC enables extraction of power from dilute bio-ethanol streams without costly concentration and purification steps. The concept should be scalable to both large and small

  2. Selection of oleaginous yeasts for fatty acid production.

    PubMed

    Lamers, Dennis; van Biezen, Nick; Martens, Dirk; Peters, Linda; van de Zilver, Eric; Jacobs-van Dreumel, Nicole; Wijffels, René H; Lokman, Christien

    2016-05-27

    Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing "waste carbon sources". In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids.

  3. 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

  4. Simulation of Yeast Cooperation in 2D.

    PubMed

    Wang, M; Huang, Y; Wu, Z

    2016-03-01

    Evolution of cooperation has been an active research area in evolutionary biology in decades. An important type of cooperation is developed from group selection, when individuals form spatial groups to prevent them from foreign invasions. In this paper, we study the evolution of cooperation in a mixed population of cooperating and cheating yeast strains in 2D with the interactions among the yeast cells restricted to their small neighborhoods. We conduct a computer simulation based on a game theoretic model and show that cooperation is increased when the interactions are spatially restricted, whether the game is of a prisoner's dilemma, snow drifting, or mutual benefit type. We study the evolution of homogeneous groups of cooperators or cheaters and describe the conditions for them to sustain or expand in an opponent population. We show that under certain spatial restrictions, cooperator groups are able to sustain and expand as group sizes become large, while cheater groups fail to expand and keep them from collapse.

  5. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  6. Low bioaccessibility of vitamin D2 from yeast-fortified bread compared to crystalline D2 bread and D3 from fluid milks.

    PubMed

    Lipkie, Tristan E; Ferruzzi, Mario G; Weaver, Connie M

    2016-11-09

    The assessment of the efficacy of dietary and supplemental vitamin D tends to be confounded by differences in the serum 25-hydroxyvitamin D response between vitamin D 2 and vitamin D 3 . Serum response differences from these vitamers may be due to differences in bioavailability. To address this specifically, the bioaccessibility was assessed for vitamin D 2 from breads fortified with UV-treated yeast, and a benchmark against staple vitamin D 3 fortified foods including bovine milks and infant formula, as well as crystalline vitamin D 2 fortified bread. Fortified foods were subjected to a three-stage static in vitro digestion model, and vitamin D was analyzed by HPLC-MS. Vitamin D bioaccessibility was significantly greater from bovine milks and infant formula (71-85%) than from yeast-fortified sandwich breads (6-7%). Bioaccessibility was not different between whole wheat and white wheat bread (p > 0.05), but was ∼4× lower from yeast-fortified bread than from crystalline vitamin D 2 fortified bread (p < 0.05). Intact yeast cells were observed in the digesta of yeast fortified bread. These results indicate that the low bioavailability of yeast D 2 in comparison to other vitamin D 2 sources is likely due to entrapment within a less digestible yeast matrix and not only to metabolic differences between vitamins D 2 and D 3 .

  7. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

    PubMed

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A

    2016-06-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3' end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. Copyright © 2016 Larson et al.

  8. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

    PubMed Central

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

    2016-01-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

  9. Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species.

    PubMed

    de Barros Lopes, Miguel; Bellon, Jennifer R; Shirley, Neil J; Ganter, Philip F

    2002-01-01

    Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.

  10. Anomalous spectral-weight transfers unraveling oxygen screening and electronic correlations in the insulator-metal transition of VO2

    NASA Astrophysics Data System (ADS)

    Yeo, L. H.; Srivastava, A.; Majidi, M. A.; Sutarto, R.; He, F.; Poh, S. M.; Diao, C.; Yu, X.; Motapothula, M.; Saha, S.; Ojha, S.; Kanjilal, D.; Trevisanutto, P. E.; Breese, M. B. H.; Venkatesan, T.; Rusydi, A.

    2015-02-01

    Vanadium dioxide (VO2) undergoes an unusual insulator-metal transition (IMT), and after decades of study, the origin of the IMT remains hotly debated. Here, by analyzing spectral-weight transfers (SWTs) of x-ray absorption spectroscopy at the V L3 ,2 and O K edges on specially designed VO2 films, we observe d||(dx2-y2) band splitting at the V L3 ,2 edges across the IMT, accompanied by anomalous SWTs as high as ˜12 eV at the O K edge, indicating strong electronic correlations. Surprisingly, a few oxygen vacancies induce dramatic SWTs at the O K edge, but the sample remains conducting. Supported by theoretical calculations, we find that in the metallic state, direct V (3 d∥) -V(3 d∥) and O(2 p ) -V(3 d∥) hybridized orbital correlations are screened by O(2 p ) -V(3 dπ) hybridized orbitals, while in the insulating state they are strongly correlated due to changes in the oxygen orbital occupancy. Our result shows the importance of screenings and electronic correlations for IMTs in VO2.

  11. Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

    PubMed

    Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

    2014-12-01

    The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

  12. From the Cover: Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

    NASA Astrophysics Data System (ADS)

    Ito, Takashi; Tashiro, Kosuke; Muta, Shigeru; Ozawa, Ritsuko; Chiba, Tomoko; Nishizawa, Mayumi; Yamamoto, Kiyoshi; Kuhara, Satoru; Sakaki, Yoshiyuki

    2000-02-01

    Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATα strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of ≈4 × 106 different combinations, constituting ≈10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

  13. Occurrence of killer yeasts in leaf-cutting ant nests.

    PubMed

    Carreiro, S C; Pagnocca, F C; Bacci, M; Bueno, O C; Hebling, M J A; Middelhoven, W J

    2002-01-01

    Killer activity was screened in 99 yeast strains isolated from the nests of the leaf-cutting ant Atta sexdens against 6 standard sensitive strains, as well as against each other. Among this yeast community killer activity was widespread since 77 strains (78%) were able to kill or inhibit the growth of at least one standard strain or nest strain. Toxin production was observed in representatives of all the studied genera including Aureobasidium, Rhodotorula, Tremella and Trichosporon, whose killer activity has not yet been described.

  14. Quantitative maps of genetic interactions in yeast - comparative evaluation and integrative analysis.

    PubMed

    Lindén, Rolf O; Eronen, Ville-Pekka; Aittokallio, Tero

    2011-03-24

    High-throughput genetic screening approaches have enabled systematic means to study how interactions among gene mutations contribute to quantitative fitness phenotypes, with the aim of providing insights into the functional wiring diagrams of genetic interaction networks on a global scale. However, it is poorly known how well these quantitative interaction measurements agree across the screening approaches, which hinders their integrated use toward improving the coverage and quality of the genetic interaction maps in yeast and other organisms. Using large-scale data matrices from epistatic miniarray profiling (E-MAP), genetic interaction mapping (GIM), and synthetic genetic array (SGA) approaches, we carried out here a systematic comparative evaluation among these quantitative maps of genetic interactions in yeast. The relatively low association between the original interaction measurements or their customized scores could be improved using a matrix-based modelling framework, which enables the use of single- and double-mutant fitness estimates and measurements, respectively, when scoring genetic interactions. Toward an integrative analysis, we show how the detections from the different screening approaches can be combined to suggest novel positive and negative interactions which are complementary to those obtained using any single screening approach alone. The matrix approximation procedure has been made available to support the design and analysis of the future screening studies. We have shown here that even if the correlation between the currently available quantitative genetic interaction maps in yeast is relatively low, their comparability can be improved by means of our computational matrix approximation procedure, which will enable integrative analysis and detection of a wider spectrum of genetic interactions using data from the complementary screening approaches.

  15. Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

    PubMed

    Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

    1986-01-01

    For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi

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

  17. Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport.

    PubMed

    Colinet, Anne-Sophie; Thines, Louise; Deschamps, Antoine; Flémal, Gaëlle; Demaegd, Didier; Morsomme, Pierre

    2017-07-01

    The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca 2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca 2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca 2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation. © 2017 John Wiley & Sons Ltd.

  18. The role of lager beer yeast in oxidative stability of model beer.

    PubMed

    Berner, T S; Arneborg, N

    2012-03-01

    In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma-mass spectrometry, and the slowest formation of radicals in the model beers. A more oxidative stable beer is not obtained by a more-oxidative-stress-tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less-oxidative-stress-tolerant strain, exhibiting a higher iron uptake. To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  19. CAVE2: a hybrid reality environment for immersive simulation and information analysis

    NASA Astrophysics Data System (ADS)

    Febretti, Alessandro; Nishimoto, Arthur; Thigpen, Terrance; Talandis, Jonas; Long, Lance; Pirtle, J. D.; Peterka, Tom; Verlo, Alan; Brown, Maxine; Plepys, Dana; Sandin, Dan; Renambot, Luc; Johnson, Andrew; Leigh, Jason

    2013-03-01

    Hybrid Reality Environments represent a new kind of visualization spaces that blur the line between virtual environments and high resolution tiled display walls. This paper outlines the design and implementation of the CAVE2TM Hybrid Reality Environment. CAVE2 is the world's first near-seamless flat-panel-based, surround-screen immersive system. Unique to CAVE2 is that it will enable users to simultaneously view both 2D and 3D information, providing more flexibility for mixed media applications. CAVE2 is a cylindrical system of 24 feet in diameter and 8 feet tall, and consists of 72 near-seamless, off-axisoptimized passive stereo LCD panels, creating an approximately 320 degree panoramic environment for displaying information at 37 Megapixels (in stereoscopic 3D) or 74 Megapixels in 2D and at a horizontal visual acuity of 20/20. Custom LCD panels with shifted polarizers were built so the images in the top and bottom rows of LCDs are optimized for vertical off-center viewing- allowing viewers to come closer to the displays while minimizing ghosting. CAVE2 is designed to support multiple operating modes. In the Fully Immersive mode, the entire room can be dedicated to one virtual simulation. In 2D model, the room can operate like a traditional tiled display wall enabling users to work with large numbers of documents at the same time. In the Hybrid mode, a mixture of both 2D and 3D applications can be simultaneously supported. The ability to treat immersive work spaces in this Hybrid way has never been achieved before, and leverages the special abilities of CAVE2 to enable researchers to seamlessly interact with large collections of 2D and 3D data. To realize this hybrid ability, we merged the Scalable Adaptive Graphics Environment (SAGE) - a system for supporting 2D tiled displays, with Omegalib - a virtual reality middleware supporting OpenGL, OpenSceneGraph and Vtk applications.

  20. A Simple and Reliable Method for Hybridization of Homothallic Wine Strains of Saccharomyces cerevisiae

    PubMed Central

    Ramírez, Manuel; Peréz, Francisco; Regodón, José A.

    1998-01-01

    A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains. PMID:9835605

  1. A Novel Hybrid Iron Regulation Network Combines Features from Pathogenic and Nonpathogenic Yeasts

    PubMed Central

    Gerwien, Franziska; Safyan, Abu; Wisgott, Stephanie; Hille, Fabrice; Kaemmer, Philipp; Linde, Jörg; Brunke, Sascha; Kasper, Lydia

    2016-01-01

    ABSTRACT Iron is an essential micronutrient for both pathogens and their hosts, which restrict iron availability during infections in an effort to prevent microbial growth. Successful human pathogens like the yeast Candida glabrata have thus developed effective iron acquisition strategies. Their regulation has been investigated well for some pathogenic fungi and in the model organism Saccharomyces cerevisiae, which employs an evolutionarily derived system. Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi. Specifically, similarly to baker’s yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes. However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens. Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi. PMID:27795405

  2. A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

    PubMed

    Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

    2014-08-01

    For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion.

  3. 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

  4. Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

    PubMed

    Vadkertiová, Renáta; Molnárová, Jana; Vránová, Dana; Sláviková, Elena

    2012-12-01

    Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

  5. Discovery of Natural Products as Novel and Potent FXR Antagonists by Virtual Screening

    NASA Astrophysics Data System (ADS)

    Diao, Yanyan; Jiang, Jing; Zhang, Shoude; Li, Shiliang; Shan, Lei; Huang, Jin; Zhang, Weidong; Li, Honglin

    2018-04-01

    Farnesoid X receptor (FXR) is a member of nuclear receptor family involved in multiple physiological processes through regulating specific target genes. The critical role of FXR as a transcriptional regulator makes it a promising target for diverse diseases, especially those related to metabolic disorders such as diabetes and cholestasis. However, the underlying activation mechanism of FXR is still a blur owing to the absence of proper FXR modulators. To identify potential FXR modulators, an in-house natural product database (NPD) containing over 4000 compounds was screened by structure-based virtual screening strategy and subsequent hit-based similarity searching method. After the yeast two-hybrid (Y2H) assay, six natural products were identified as FXR antagonists which blocked the CDCA-induced SRC-1 association. The IC50 values of compounds 2a, a diterpene bearing polycyclic skeleton, and 3a, named daphneone with chain scaffold, are as low as 1.29 μM and 1.79 μM, respectively. Compared to the control compound guggulsterone (IC50 = 6.47 μM), compounds 2a and 3a displayed 5-fold and 3-fold higher antagonistic activities against FXR, respectively. Remarkably, the two representative compounds shared low topological similarities with other reported FXR antagonists. According to the putative binding poses, the molecular basis of these antagonists against FXR was also elucidated in this report.

  6. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    PubMed Central

    Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

  7. Visualization and Image Analysis of Yeast Cells.

    PubMed

    Bagley, Steve

    2016-01-01

    When converting real-life data via visualization to numbers and then onto statistics the whole system needs to be considered so that conversion from the analogue to the digital is accurate and repeatable. Here we describe the points to consider when approaching yeast cell analysis visualization, processing, and analysis of a population by screening techniques.

  8. Genome-wide bisulfite sensitivity profiling of yeast suggests bisulfite inhibits transcription.

    PubMed

    Segovia, Romulo; Mathew, Veena; Tam, Annie S; Stirling, Peter C

    2017-09-01

    Bisulfite, in the form of sodium bisulfite or metabisulfite, is used commercially as a food preservative. Bisulfite is used in the laboratory as a single-stranded DNA mutagen in epigenomic analyses of DNA methylation. Recently it has also been used on whole yeast cells to induce mutations in exposed single-stranded regions in vivo. To understand the effects of bisulfite on live cells we conducted a genome-wide screen for bisulfite sensitive mutants in yeast. Screening the deletion mutant array, and collections of essential gene mutants we define a genetic network of bisulfite sensitive mutants. Validation of screen hits revealed hyper-sensitivity of transcription and RNA processing mutants, rather than DNA repair pathways and follow-up analyses support a role in perturbation of RNA transactions. We propose a model in which bisulfite-modified nucleotides may interfere with transcription or RNA metabolism when used in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products.

    PubMed

    Ogunremi, O R; Sanni, A I; Agrawal, R

    2015-09-01

    To determine the starter culture and multifunctional potentials of yeast strains from some cereal-based Nigerian traditional fermented food products. Yeast isolates were screened for enzyme production and identified by sequencing the D1/D2 region of 26S rDNA. Pichia kluyveri LKC17, Issatchenkia orientalis OSL11, Pichia kudriavzevii OG32, Pichia kudriavzevii ROM11 and Candida tropicalis BOM21 exhibited the highest protease, lipase and phytase activity. They were selected and further evaluated for gastrointestinal survival and adherence ability. Although strain-specific, they retained viability at 37°C and showed survival at pH 2·0., I. orientalis OSL11 showed the highest survival at 2% bile salts concentration and P. kudriavzevii ROM11 showed the least survival. The yeast strains showed strong autoaggregation ability (81·24-91·85%) and hydrophobicity to n-hexadecane (33·61-42·30%). The highest co-aggregation ability was detected for P. kudriavzevii OG32 and Escherichia coli (71·57%). All the yeast strains removed cholesterol in the range of 49·03-74·05% over 48 h and scavenged for free radicals in methanol reaction system. In this study, we isolated new yeast strains with multifunctional potentials that can be used as functional starter cultures to produce cereal-based probiotic products. The development of probiotic yeast strains as starter culture to improve the quality attributes and confer functional value on cereal-based traditional fermented foods is beneficial. © 2015 The Society for Applied Microbiology.

  10. Physiological and genomic characterisation of Saccharomyces cerevisiae hybrids with improved fermentation performance and mannoprotein release capacity.

    PubMed

    Pérez-Través, Laura; Lopes, Christian A; González, Ramón; Barrio, Eladio; Querol, Amparo

    2015-07-16

    Yeast mannoproteins contribute to several aspects of wine quality by protecting wine against protein haze, reducing astringency, retaining aroma compounds and stimulating lactic-acid bacteria growth. The selection of a yeast strain that simultaneously overproduces mannoproteins and presents good fermentative characteristics is a difficult task. In this work, a Saccharomyces cerevisiae×S. cerevisiae hybrid bearing the two oenologically relevant features was constructed. According to the genomic characterisation of the hybrids, different copy numbers of some genes probably related with these physiological features were detected. The hybrid shared not only a similar copy number of genes SPR1, SWP1, MNN10 and YPS7 related to cell wall integrity with parental Sc1, but also a similar copy number of some glycolytic genes with parental Sc2, such as GPM1 and HXK1, as well as the genes involved in hexose transport, such as HXT9, HXT11 and HXT12. This work demonstrates that hybridisation and stabilisation under winemaking conditions constitute an effective approach to obtain yeast strains with desirable physiological features, like mannoprotein overproducing capacity and improved fermentation performance, which genetically depend of the expression of numerous genes (multigenic characters). Copyright © 2015. Published by Elsevier B.V.

  11. Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study.

    PubMed

    Yang, Zhihong; Lin, James; Zhang, John; Fong, Wai Ieng; Li, Pei; Zhao, Rong; Liu, Xiaohong; Podevin, William; Kuang, Yanping; Liu, Jiaen

    2015-06-23

    Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening. This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups. NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05). While NGS screening

  12. The Cak1p Protein Kinase Is Required at G(1)/S and G(2)/M in the Budding Yeast Cell Cycle

    PubMed Central

    Sutton, A.; Freiman, R.

    1997-01-01

    The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G(2) to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G(2) to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G(1) into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function. PMID:9286668

  13. Alcoholic fermentation of d-xylose by yeasts. [Brettanomyces naardenensis; Candida shehatae; Candida tenuis; Pachysolen tannaphilus, Pichia segobiensis; Pichia stipitis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Toivola, A.; Yarrow, D.; van den Bosch, E.

    1984-06-01

    Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of D-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment D-cellobiose revealed that only Candida tenuis CBS 4435 was a goodmore » fermenter of both xylose and cellobiose under the test conditions used.« less

  14. Fabrication of monolayer MoS2/rGO hybrids with excellent tribological performances through a surfactant-assisted hydrothermal route

    NASA Astrophysics Data System (ADS)

    Chen, Jinsuo; Xia, Yunfei; Yang, Jin; Chen, Beibei

    2018-06-01

    The extremely low friction between incommensurate two-dimensional (2D) atomic layers has recently attracted a great interest. Here, we demonstrated a promising surfactant-assisted strategy for the synthesis of MoS2/reduced graphene oxide (MoS2/rGO) hybrid materials with monolayer MoS2 and rGO, which exhibited excellent tribological metrics with a friction coefficient of ˜ 0.09 and a wear rate of ˜ 2.08 × 10-5 mm3/Nm in the ethanol dispersion. The incommensurate 2D atomic layer interface formed due to intrinsic lattice mismatch between MoS2 and graphene was thought to be responsible for the excellent lubricating performances. In addition to the benefits of unique hybrid structure, MoS2/rGO hybrids could also adsorb on metal surfaces and screen the metal-metal interaction to passivate the metal surfaces with a consequent reduction of corrosion wear during sliding. This work could pave a new pathway to design novel materials for pursuing excellent tribological properties by hybridizing different 2D atomic-layered materials.

  15. A diversity study of Saccharomycopsis fibuligera in rice wine starter nuruk, reveals the evolutionary process associated with its interspecies hybrid.

    PubMed

    Farh, Mohamed El-Agamy; Cho, Yunjoo; Lim, Jae Yun; Seo, Jeong-Ah

    2017-05-01

    The amylolytic yeast Saccharomycopsis fibuligera is the predominant yeast in the starter product, nuruk, which is utilized for rice wine production in South Korea. Latest molecular studies explore a recently developed interspecific hybridization among stains of S. fibuligera with a unique genetic feature. However, the origin of the natural hybridization occurrence is still unclear. Thus, to respectively distinguish parental and hybrid strains, specific primer sets were applied on 141 yeast strains isolated from different nuruk samples fermented in different provinces. Sixty-seven strains were defined accordingly as parental species with genome A while 8 strains were defined as hybrid strains. Unexpectedly, another parental species with genome B could not be found among the strain pools yet. Furthermore, it was observed that hybrid strains are phenotypically different from A genome strains; asci containing tetrad ascospores were observed in A genome strains more frequent than in hybrid strains. Nevertheless, hybrid strains were slightly more thermotolerant than A genome strains. Interestingly, all hybrid strains were located only in Jeju province. Based on these sets of data, we speculated that the unique climate of Jeju province might play an evolutionary role in the interspecific hybridization between A genome strains, as well as the unculturable allopatric B genome strains.

  16. 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

  17. Increased mannoprotein content in wines produced by Saccharomyces kudriavzevii×Saccharomyces cerevisiae hybrids.

    PubMed

    Pérez-Través, Laura; Querol, Amparo; Pérez-Torrado, Roberto

    2016-11-21

    Several wine quality aspects are influenced by yeast mannoproteins on account of aroma compounds retention, lactic-acid bacterial growth stimulation, protection against protein haze and astringency reduction. Thus selecting a yeast strain that produces high levels of mannoproteins is important for the winemaking industry. In this work, we observed increased levels of mannoproteins in S. cerevisiae×S. kudriavzevii hybrids, compared to the S. cerevisiae strain, in wine fermentations. Furthermore, the expression of a key gene related to mannoproteins biosynthesis, PMT1, increased in the S. cerevisiae×S. kudriavzevii hybrid. We showed that artificially constructed S. cerevisiae×S. kudriavzevii hybrids also increased the levels of mannoproteins. This work demonstrates that either natural or artificial S. cerevisiae×S. kudriavzevii hybrids present mannoprotein overproducing capacity under winemaking conditions, a desirable physiological feature for this industry. These results suggest that genome interaction in hybrids generates a physiological environment that enhances the release of mannoproteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Synthetic Genetic Arrays: Automation of Yeast Genetics.

    PubMed

    Kuzmin, Elena; Costanzo, Michael; Andrews, Brenda; Boone, Charles

    2016-04-01

    Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype. © 2016 Cold Spring Harbor Laboratory Press.

  19. Breeding of Freeze-tolerant Yeast and the Mechanisms of Stress-tolerance

    NASA Astrophysics Data System (ADS)

    Hino, Akihiro

    Frozen dough method have been adopted in the baking industry to reduce labor and to produce fresh breads in stores. New freeze-tolerant yeasts for frozen dough preparations were isolated from banana peel and identified. To obtain strains that have fermentative ability even after several months of frozen storage in fermented dough, we attempted to breed new freeze-tolerantstrain. The hybrid between S.cerevisiae, which is a isolated freeze-tolerant strain, and a strain isolated from bakers' yeast with sexual conjugation gave a good quality bread made from frozen dough method. Freeze-tolerant strains showed higher surviving and trehalose accumulating abilities than freeze-sensitive strains. The freeze tolerance of the yeasts was associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period. The complicated metabolic pathway and the regulation system of trehalose in yeast cells are introduced. The trehalose synthesis may act as a metabolic buffer system which contribute to maintain the intracellular inorganic phosphate and as a feedback regulation system in the glycolysis. However, it is not known enough how the trehalose protects yeast cells from stress.

  20. Primary cervical cancer screening with human papillomavirus: end of study results from the ATHENA study using HPV as the first-line screening test.

    PubMed

    Wright, Thomas C; Stoler, Mark H; Behrens, Catherine M; Sharma, Abha; Zhang, Guili; Wright, Teresa L

    2015-02-01

    ATHENA evaluated the cobas HPV Test as the primary screen for cervical cancer in women ≥25years. This reports the 3-year end-of-study results comparing the performance of HPV primary screening to different screening and triage combinations. 42,209 women ≥25years were enrolled and had cytology and hrHPV testing. Women with abnormal cytology (≥atypical squamous cells of undetermined significance) and those HPV positive were referred to colposcopy. Women not reaching the study endpoint of CIN2+ entered the 3-year follow-up phase. 3-year CIR of CIN3+ in cytology-negative women was 0.8% (95% CI; 0.5-1.1%), 0.3% (95% CI 0.1-0.7%) in HPV-negative women, and 0.3% (95% CI; 0.1-0.6%) in cytology and HPV negative women. The sensitivity for CIN3+ of cytology was 47.8% (95% CI; 41.6-54.1%) compared to 61.7% (95% CI; 56.0-67.5%) for the hybrid strategy (cytology if 25-29years and cotesting with cytology and HPV if ≥30years) and 76.1% (95% CI; 70.3-81.8%) for HPV primary. The specificity for CIN3+ was 97.1% (95% CI; 96.9-97.2%), 94.6% (95% CI; 94.4-94.8%), and 93.5% (95% CI; 93.3-93.8%) for cytology, hybrid strategy, and HPV primary, respectively. Although HPV primary detects significantly more cases of CIN3+ in women ≥25years than either cytology or hybrid strategy, it requires significantly more colposcopies. However, the number of colposcopies required to detect a single CIN3+ is the same as for the hybrid strategy. HPV primary screening in women ≥25years is as effective as a hybrid screening strategy that uses cytology if 25-29years and cotesting if ≥30years. However, HPV primary screening requires less screening tests. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. The development of an unconventional food regeneration process: Quantifying the nutritional components of a model methylotrophic yeast

    NASA Technical Reports Server (NTRS)

    Petersen, G. R.; Stokes, B. O.

    1986-01-01

    A hybrid chemical/biological approach to unconventional food regeneration is discussed. Carbon dioxide and water, the major wastes of human metabolism would be converted to methanol by one of several physiochemical processes available (thermal, photocatalytic, etc.). Methanol is then used to supply carbon and energy for the culture of microorganisms which in turn produce biological useful basic food stuffs for human nutrition. Our work has focused on increasing the carbohydrate levels of a candidate methylotrophic yeast to more nearly coincide with human nutritional requirements. Yeasts were chosen due to their high carbohydrate levels compared to bacteria and their present familiarity in the human diet. The initial candidate yeast studied was a thermotolerant strain of Hansenula polymor pha, DL-1. The quantitative results that permit an evaluation of the overall efficiency in hybrid chemical/biological food production schemes are discussed. A preliminary evaluation of the overall efficiency of such schemes is also discussed.

  2. Efficient activation of transcription in yeast by the BPV1 E2 protein.

    PubMed Central

    Stanway, C A; Sowden, M P; Wilson, L E; Kingsman, A J; Kingsman, S M

    1989-01-01

    The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level. Images PMID:2539584

  3. (GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

    PubMed

    Ramírez-Castrillón, Mauricio; Mendes, Sandra Denise Camargo; Inostroza-Ponta, Mario; Valente, Patricia

    2014-01-01

    In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

  4. (GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?

    PubMed Central

    Inostroza-Ponta, Mario; Valente, Patricia

    2014-01-01

    In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure. PMID:25171185

  5. Chemical signaling and insect attraction is a conserved trait in yeasts.

    PubMed

    Becher, Paul G; Hagman, Arne; Verschut, Vasiliki; Chakraborty, Amrita; Rozpędowska, Elżbieta; Lebreton, Sébastien; Bengtsson, Marie; Flick, Gerhard; Witzgall, Peter; Piškur, Jure

    2018-03-01

    Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae , the insect-associated species Candida californica , Pichia kluyveri and Metschnikowia andauensis , wine yeast Dekkera bruxellensis , milk yeast Kluyveromyces lactis , the vertebrate pathogens Candida albicans and Candida glabrata , and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co-occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila , we tested the basal hexapod Folsomia candida (Collembola) in a Y-tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts

  6. Mapping replication origins in yeast chromosomes.

    PubMed

    Brewer, B J; Fangman, W L

    1991-07-01

    The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

  7. Screening and identification of host factors interacting with UL14 of herpes simplex virus 1.

    PubMed

    Wu, Fuqing; Xing, Junji; Wang, Shuai; Li, Meili; Zheng, Chunfu

    2011-08-01

    The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

  8. K2 killer toxin-induced physiological changes in the yeast Saccharomyces cerevisiae.

    PubMed

    Orentaite, Irma; Poranen, Minna M; Oksanen, Hanna M; Daugelavicius, Rimantas; Bamford, Dennis H

    2016-03-01

    Saccharomyces cerevisiae cells produce killer toxins, such as K1, K2 and K28, that can modulate the growth of other yeasts giving advantage for the killer strains. Here we focused on the physiological changes induced by K2 toxin on a non-toxin-producing yeast strain as well as K1, K2 and K28 killer strains. Potentiometric measurements were adjusted to observe that K2 toxin immediately acts on the sensitive cells leading to membrane permeability. This correlated with reduced respiration activity, lowered intracellular ATP content and decrease in cell viability. However, we did not detect any significant ATP leakage from the cells treated by killer toxin K2. Strains producing heterologous toxins K1 and K28 were less sensitive to K2 than the non-toxin producing one suggesting partial cross-protection between the different killer systems. This phenomenon may be connected to the observed differences in respiratory activities of the killer strains and the non-toxin-producing strain at low pH. This might also have practical consequences in wine industry; both as beneficial ones in controlling contaminating yeasts and non-beneficial ones causing sluggish fermentation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Enterovirus 71 2C Protein Inhibits NF-κB Activation by Binding to RelA(p65)

    PubMed Central

    Du, Haiwei; Yin, Peiqi; Yang, Xiaojie; Zhang, Leiliang; Jin, Qi; Zhu, Guofeng

    2015-01-01

    Viruses evolve multiple ways to interfere with NF-κB signaling, a key regulator of innate and adaptive immunity. Enterovirus 71 (EV71) is one of primary pathogens that cause hand-foot-mouth disease. Here, we identify RelA(p65) as a novel binding partner for EV71 2C protein from yeast two-hybrid screen. By interaction with IPT domain of p65, 2C reduces the formation of heterodimer p65/p50, the predominant form of NF-κB. We also show that picornavirus 2C family proteins inhibit NF-κB activation and associate with p65 and IKKβ. Our findings provide a novel mechanism how EV71 antagonizes innate immunity. PMID:26394554

  10. Overexpression of the transcription activator Msn2 enhances the fermentation ability of industrial baker's yeast in frozen dough.

    PubMed

    Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

    2012-01-01

    We constructed a self-cloning diploid baker's yeast strain that overexpressed the transcription activator Msn2. It showed higher tolerance to freeze-thaw stress and higher intracellular trehalose level than observed in the wild-type strain. Overexpression of Msn2 also enhanced the fermentation ability of baker's yeast cells in frozen dough. Hence, Msn2-overexpressing baker's yeast should be useful in frozen-dough baking.

  11. New Trends in the Uses of Yeasts in Oenology.

    PubMed

    Querol, Amparo; Pérez-Torrado, Roberto; Alonso-Del-Real, Javier; Minebois, Romain; Stribny, Jiri; Oliveira, Bruno M; Barrio, Eladio

    2018-01-01

    The most important factor in winemaking is the quality of the final product and the new trends in oenology are dictated by wine consumers and producers. Traditionally the red wine is the most consumed and more popular; however, in the last times, the wine companies try to attract other groups of populations, especially young people and women that prefer sweet, whites or rosé wines, very fruity and with low alcohol content. Besides the new trends in consumer preferences, there are also increased concerns on the effects of alcohol consumption on health and the effects of global climate change on grape ripening and wine composition producing wines with high alcohol content. Although S. cerevisiae is the most frequent species in wines, and the subject of most studies, S. uvarum and hybrids between Saccharomyces species such as S. cerevisiae×S. kudriavzevii and S. cerevisiae×S. uvarum are also involved in wine fermentations and can be preponderant in certain wine regions. New yeast starters of non-cerevisiae strains (S. uvarum) or hybrids (S. cerevisiae×S. uvarum and S. cerevisiae×S. kudriavzevii) can contribute to solve some problems of the wineries. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts, while fulfilling the requirements of the commercial yeasts, such as a good fermentative performance and aromatic profiles that are of great interest for the wine industry. In this review, we will analyze different applications of nonconventional yeasts to solve the current winemaking demands. © 2018 Elsevier Inc. All rights reserved.

  12. Monitoring Recombination During Meiosis in Budding Yeast.

    PubMed

    Owens, Shannon; Tang, Shangming; Hunter, Neil

    2018-01-01

    Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization. © 2018 Elsevier Inc. All rights reserved.

  13. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    PubMed

    Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

    2012-01-01

    The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  14. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

    PubMed Central

    Helsdingen, Richard J. R.; Hamers, Astrid R. M.; van Duursen, Majorie B. M.; Nielen, Michel W. F.; Hoogenboom, Ron L. A. P.

    2007-01-01

    Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action. PMID:17849102

  15. Continuous-flow separation of live and dead yeasts using reservoir-based dielectrophoresis (rDEP)

    NASA Astrophysics Data System (ADS)

    Patel, Saurin; Showers, Daniel; Vedantam, Pallavi; Tzeng, Tzuen-Rong; Qian, Shizhi; Xuan, Xiangchun

    2012-11-01

    Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening etc. We develop a novel microfluidic approach to continuous separation of yeast cells by viability inside a reservoir. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeasts and continuously sort them from live ones. We term this approach reservoir-based dielectrophoresis (rDEP). The transporting, focusing, and trapping of live and dead yeast cells at the reservoir-microchannel junction are studied separately by varying the DC-biased AC electric fields. These phenomena can all be reasonably predicted by a 2D numerical model. We find that the AC to DC field ratio for live yeast trapping is higher than that for dead cells because the former experiences a weaker rDEP while having a larger electrokinetic mobility. It is this difference in the AC to DC field ratio that enables the viability-based yeast cell separation. The rDEP approach has unique advantages over existing DEP-based techniques such as the occupation of zero channel space and the elimination of in-channel mechanical or electrical parts. NSF

  16. Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

    PubMed

    Navarro-Arias, María J; Dementhon, Karine; Defosse, Tatiana A; Foureau, Emilien; Courdavault, Vincent; Clastre, Marc; Le Gal, Solène; Nevez, Gilles; Le Govic, Yohann; Bouchara, Jean-Philippe; Giglioli-Guivarc'h, Nathalie; Noël, Thierry; Mora-Montes, Hector M; Papon, Nicolas

    2017-09-01

    Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  17. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

    PubMed

    Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

    2016-12-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.

  18. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability

    PubMed Central

    Ball, David A.

    2016-01-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally. PMID:27935947

  19. 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

  20. Strategies for identifying new prions in yeast

    PubMed Central

    MacLea, Kyle S

    2011-01-01

    The unexpected discovery of two prions, [URE3] and [PSI+], in Saccharomyces cerevisiae led to questions about how many other proteins could undergo similar prion-based structural conversions. However, [URE3] and [PSI+] were discovered by serendipity in genetic screens. Cataloging the full range of prions in yeast or in other organisms will therefore require more systematic search methods. Taking advantage of some of the unique features of prions, various researchers have developed bioinformatic and experimental methods for identifying novel prion proteins. These methods have generated long lists of prion candidates. The systematic testing of some of these prion candidates has led to notable successes; however, even in yeast, where rapid growth rate and ease of genetic manipulation aid in testing for prion activity, such candidate testing is laborious. Development of better methods to winnow the field of prion candidates will greatly aid in the discovery of new prions, both in yeast and in other organisms, and help us to better understand the role of prions in biology. PMID:22052351

  1. Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

    PubMed

    Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

    2016-09-01

    Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

  2. Mating of 2 Laboratory Saccharomyces cerevisiae Strains Resulted in Enhanced Production of 2-Phenylethanol by Biotransformation of L-Phenylalanine.

    PubMed

    Mierzejewska, Jolanta; Tymoszewska, Aleksandra; Chreptowicz, Karolina; Krol, Kamil

    2017-01-01

    2-Phenylethanol (2-PE) is an aromatic alcohol with a rosy scent which is widely used in the food, fragrance, and cosmetic industries. Promising sources of natural 2-PE are microorganisms, especially yeasts, which can produce 2-PE by biosynthesis and biotransformation. Thus, the first challenging goal in the development of biotechnological production of 2-PE is searching for highly productive yeast strains. In the present work, 5 laboratory Saccharomyces cerevisiae strains were tested for the production of 2-PE. Thereafter, 2 of them were hybridized by a mating procedure and, as a result, a new diploid, S. cerevisiae AM1-d, was selected. Within the 72-h batch culture in a medium containing 5 g/L of L-phenylalanine, AM1-d produced 3.83 g/L of 2-PE in a shaking flask. In this way, we managed to select the diploid S. cerevisiae AM1-d strain, showing a 3- and 5-fold increase in 2-PE production in comparison to parental strains. Remarkably, the enhanced production of 2-PE by the hybrid of 2 yeast laboratory strains is demonstrated here for the first time. © 2017 S. Karger AG, Basel.

  3. A Yeast Model of FUS/TLS-Dependent Cytotoxicity

    PubMed Central

    Ju, Shulin; Tardiff, Daniel F.; Han, Haesun; Divya, Kanneganti; Zhong, Quan; Maquat, Lynne E.; Bosco, Daryl A.; Hayward, Lawrence J.; Brown, Robert H.; Lindquist, Susan; Ringe, Dagmar; Petsko, Gregory A.

    2011-01-01

    FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. PMID:21541368

  4. Yeast proteome map (last update).

    PubMed

    Perrot, Michel; Moes, Suzette; Massoni, Aurélie; Jenoe, Paul; Boucherie, Hélian

    2009-10-01

    The identification of proteins separated on 2-D gels is essential to exploit the full potential of 2-D gel electrophoresis for proteomic investigations. For this purpose we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification by mass spectrometry of 100 novel yeast protein spots that have so far not been tackled due to their scarcity on our standard 2-D gels. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 716. They correspond to 485 unique proteins. Among these, 154 were resolved into several isoforms. The present data set can now be expanded to report for the first time a map of 363 protein isoforms that significantly deepens our knowledge of the yeast proteome. The reference map and a list of all identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).

  5. Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants.

    PubMed

    Rodrigues, Andre; Cable, Rachel N; Mueller, Ulrich G; Bacci, Maurício; Pagnocca, Fernando C

    2009-10-01

    We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.

  6. Effects of Malassezia yeasts on serum Th1 and Th2 cytokines in patients with guttate psoriasis.

    PubMed

    Aydogan, Kenan; Tore, Okan; Akcaglar, Sevim; Oral, Barbaros; Ener, Beyza; Tunalı, Sukran; Saricaoglu, Hayriye

    2013-01-01

    Systemic and focal infections caused by microorganisms have been known to induce or exacerbate psoriasis. Although the role of yeast species of the genus Malassezia in the pathogenesis of psoriasis is not fully understood, it is thought that these lipophilic yeasts may represent a triggering factor in the exacerbation of psoriatic lesions. This study investigated the effects of Malassezia yeasts on serum Th1 and Th2 cytokines in patients with guttate psoriasis (GP) in order to define their role in the pathogenesis of psoriasis. Fifty patients with GP and 29 clinically healthy individuals were included in the study. All samples consisted of scales and scrapings taken from the scalps, trunks, and upper limbs of both psoriasis patients and healthy subjects. Psoriasis patients and healthy subjects were grouped according to their positivity or negativity for Malassezia yeasts as ascertained by direct microscopy and/or culture. An enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of Th1 and Th2 cytokines in these groups. No significant differences in positivity for Malassezia yeasts were found between psoriatic skin and healthy skin in samples taken from different body sites. Serum interleukin-13 (IL-13) levels were significantly lower in the psoriasis group compared with the control group (P = 0.04). Levels of other cytokines did not differ significantly between the psoriasis and control groups. Mean levels of Th2 cytokines (IL-4, IL-10, IL-13), but not of Th1 cytokines (IL-2 and IFN-γ), were significantly lower in psoriasis patients positive for Malassezia yeasts compared with those negative for Malassezia yeasts and control subjects (P = 0.04, P < 0.001 and P = 0.01, respectively). The isolation of Malassezia yeasts from GP lesions does not necessarily mean that these species are pathogenic, but their downregulating effects on anti-inflammatory Th2 cytokines may contribute to the occurrence of GP. © 2012 The International Society of

  7. Combining structure-based pharmacophore modeling, virtual screening, and in silico ADMET analysis to discover novel tetrahydro-quinoline based pyruvate kinase isozyme M2 activators with antitumor activity

    PubMed Central

    Chen, Can; Wang, Ting; Wu, Fengbo; Huang, Wei; He, Gu; Ouyang, Liang; Xiang, Mingli; Peng, Cheng; Jiang, Qinglin

    2014-01-01

    Compared with normal differentiated cells, cancer cells upregulate the expression of pyruvate kinase isozyme M2 (PKM2) to support glycolytic intermediates for anabolic processes, including the synthesis of nucleic acids, amino acids, and lipids. In this study, a combination of the structure-based pharmacophore modeling and a hybrid protocol of virtual screening methods comprised of pharmacophore model-based virtual screening, docking-based virtual screening, and in silico ADMET (absorption, distribution, metabolism, excretion and toxicity) analysis were used to retrieve novel PKM2 activators from commercially available chemical databases. Tetrahydroquinoline derivatives were identified as potential scaffolds of PKM2 activators. Thus, the hybrid virtual screening approach was applied to screen the focused tetrahydroquinoline derivatives embedded in the ZINC database. Six hit compounds were selected from the final hits and experimental studies were then performed. Compound 8 displayed a potent inhibitory effect on human lung cancer cells. Following treatment with Compound 8, cell viability, apoptosis, and reactive oxygen species (ROS) production were examined in A549 cells. Finally, we evaluated the effects of Compound 8 on mice xenograft tumor models in vivo. These results may provide important information for further research on novel PKM2 activators as antitumor agents. PMID:25214764

  8. Insight into the Role of Ca2+-Binding Protein 5 in Vesicle Exocytosis

    PubMed Central

    Sokal, Izabela

    2011-01-01

    Purpose. CaBP5 is a neuronal calmodulin-like Ca2+-binding protein that is expressed in the retina and in the cochlea. Although CaBP5 knockout mice displayed reduced sensitivity of retinal ganglion cell light responses, the function of CaBP5 in vivo is still unknown. To gain further insight into CaBP5 function, the authors screened for CaBP5-interacting partners. Methods. Potential retinal interacting partners for CaBP5 were identified using affinity chromatography followed by mass spectrometry and by yeast two-hybrid screening of a bovine retina cDNA library. Interacting partners were further analyzed using coimmunoprecipitation. Immunohistochemistry and subcellular fractionation were performed to determine their colocalization in the retina. The effect of CaBP5 on dopamine release and neurite outgrowth of PC12 cells was analyzed using ELISA and fluorescent labeling. Results. Using affinity chromatography, the authors identified Munc18–1 and myosin VI as interacting partners for CaBP5. Munc18–1 was also identified using the yeast two-hybrid system. Colocalization and coimmunoprecipitation of CaBP5 with these two proteins in retinal tissue further established their physiological interactions. Furthermore, CaBP5 expression in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis. Conclusions. This study shows that CaBP5 interacts with Munc18–1 and myosin VI, two proteins involved in the synaptic vesicle cycle. Together with the effect of CaBP5 in stimulating neurite outgrowth and vesicle exocytosis in PC12 cells, these results suggest that CaBP5 plays a role in neurotransmitter release. PMID:22039235

  9. Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains.

    PubMed

    Kong, In Iok; Turner, Timothy Lee; Kim, Heejin; Kim, Soo Rin; Jin, Yong-Su

    2018-02-01

    Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast. © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Comparative Lipidomic Profiling of S. cerevisiae and Four Other Hemiascomycetous Yeasts

    PubMed Central

    Hein, Eva-Maria; Hayen, Heiko

    2012-01-01

    Glycerophospholipids (GP) are the building blocks of cellular membranes and play essential roles in cell compartmentation, membrane fluidity or apoptosis. In addition, GPs are sources for multifunctional second messengers. Whereas the genome and proteome of the most intensively studied eukaryotic model organism, the baker’s yeast (Saccharomyces cerevisiae), are well characterized, the analysis of its lipid composition is still at the beginning. Moreover, different yeast species can be distinguished on the DNA, RNA and protein level, but it is currently unknown if they can also be differentiated by determination of their GP pattern. Therefore, the GP compositions of five different yeast strains, grown under identical environmental conditions, were elucidated using high performance liquid chromatography coupled to negative electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry in single and multistage mode. Using this approach, relative quantification of more than 100 molecular species belonging to nine GP classes was achieved. The comparative lipidomic profiling of Saccharomyces cerevisiae, Saccharomyces bayanus, Kluyveromyces thermotolerans, Pichia angusta, and Yarrowia lipolytica revealed characteristic GP profiles for each strain. However, genetically related yeast strains show similarities in their GP compositions, e.g., Saccharomyces cerevisiae and Saccharomyces bayanus. PMID:24957378

  11. Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol.

    PubMed

    Lee, Cho-Ryong; Sung, Bong Hyun; Lim, Kwang-Mook; Kim, Mi-Jin; Sohn, Min Jeong; Bae, Jung-Hoon; Sohn, Jung-Hoon

    2017-06-30

    To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6-2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

  12. Navigating yeast genome maintenance with functional genomics.

    PubMed

    Measday, Vivien; Stirling, Peter C

    2016-03-01

    Maintenance of genome integrity is a fundamental requirement of all organisms. To address this, organisms have evolved extremely faithful modes of replication, DNA repair and chromosome segregation to combat the deleterious effects of an unstable genome. Nonetheless, a small amount of genome instability is the driver of evolutionary change and adaptation, and thus a low level of instability is permitted in populations. While defects in genome maintenance almost invariably reduce fitness in the short term, they can create an environment where beneficial mutations are more likely to occur. The importance of this fact is clearest in the development of human cancer, where genome instability is a well-established enabling characteristic of carcinogenesis. This raises the crucial question: what are the cellular pathways that promote genome maintenance and what are their mechanisms? Work in model organisms, in particular the yeast Saccharomyces cerevisiae, has provided the global foundations of genome maintenance mechanisms in eukaryotes. The development of pioneering genomic tools inS. cerevisiae, such as the systematic creation of mutants in all nonessential and essential genes, has enabled whole-genome approaches to identifying genes with roles in genome maintenance. Here, we review the extensive whole-genome approaches taken in yeast, with an emphasis on functional genomic screens, to understand the genetic basis of genome instability, highlighting a range of genetic and cytological screening modalities. By revealing the biological pathways and processes regulating genome integrity, these analyses contribute to the systems-level map of the yeast cell and inform studies of human disease, especially cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. Screened Hybrid and DFT + U Studies of the Structural, Electronic, and Optical Properties of U3O8

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wen, Xiaodong; Martin, Richard L.; Scuseria, Gustavo E.

    2012-11-26

    A systematic comparison of the structures and electronic and optical properties of U3O8 in the c2mm, P¯62m, and P21/m structures (the α, β, and γ phases, respectively) is performed using density functional theory + U (PBE + U) and the Heyd–Scuseria–Ernzerhof screened hybrid functional (HSE). The relationship between the semiconducting C2mm phase of U3O8 and the high temperature, metallic P¯62m phase is explored in more detail. Our calculated results show that the HSE functional gives a better description of the electronic and optical properties when compared with available experimental data for the α and β phases, but neither approach doesmore » particularly well for the high pressure γ phase.« less

  14. Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

    PubMed

    Aung, Hsu Mon; Huangteerakul, Chananya; Panvongsa, Wittaya; Jensen, Amornrat N; Chairoungdua, Arthit; Sukrong, Suchada; Jensen, Laran T

    2018-09-15

    Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF). Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

    PubMed

    Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

    2016-10-01

    Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

  16. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

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

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

  17. Reconstruction of the evolutionary history of Saccharomyces cerevisiae x S. kudriavzevii hybrids based on multilocus sequence analysis.

    PubMed

    Peris, David; Lopes, Christian A; Arias, Armando; Barrio, Eladio

    2012-01-01

    In recent years, interspecific hybridization and introgression are increasingly recognized as significant events in the evolution of Saccharomyces yeasts. These mechanisms have probably been involved in the origin of novel yeast genotypes and phenotypes, which in due course were to colonize and predominate in the new fermentative environments created by human manipulation. The particular conditions in which hybrids arose are still unknown, as well as the number of possible hybridization events that generated the whole set of natural hybrids described in the literature during recent years. In this study, we could infer at least six different hybridization events that originated a set of 26 S. cerevisiae x S. kudriavzevii hybrids isolated from both fermentative and non-fermentative environments. Different wine S. cerevisiae strains and European S. kudriavzevii strains were probably involved in the hybridization events according to gene sequence information, as well as from previous data on their genome composition and ploidy. Finally, we postulate that these hybrids may have originated after the introduction of vine growing and winemaking practices by the Romans to the present Northern vine-growing limits and spread during the expansion of improved viticulture and enology practices that occurred during the Late Middle Ages.

  18. Analysis of estrogenic activity in environmental waters in Rio de Janeiro state (Brazil) using the yeast estrogen screen.

    PubMed

    Dias, Amanda Cristina Vieira; Gomes, Frederico Wegenast; Bila, Daniele Maia; Sant'Anna, Geraldo Lippel; Dezotti, Marcia

    2015-10-01

    The estrogenicity of waters collected from an important hydrological system in Brazil (Paraiba do Sul and Guandu Rivers) was assessed using the yeast estrogen screen (YES) assay. Sampling was performed in rivers and at the outlets of conventional water treatment plants (WTP). The removal of estrogenic activity by ozonation and chlorination after conventional water treatment (clarification and sand filtration) was investigated employing samples of the Guandu River spiked with estrogens and bisphenol A (BPA). The results revealed a preoccupying incidence of estrogenic activity at levels higher than 1ngL(-1) along some points of the rivers. Another matter of concern was the number of samples from WTPs presenting estrogenicity surpassing 1ngL(-1). The oxidation techniques (ozonation and chlorination) were effective for the removal of estrogenic activity and the combination of both techniques led to good results using less amounts of oxidants. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Yeast Derived LysA2 Can Control Bacterial Contamination in Ethanol Fermentation.

    PubMed

    Kim, Jun-Seob; Daum, M Angela; Jin, Yong-Su; Miller, Michael J

    2018-05-24

    Contamination of fuel-ethanol fermentations continues to be a significant problem for the corn and sugarcane-based ethanol industries. In particular, members of the Lactobacillaceae family are the primary bacteria of concern. Currently, antibiotics and acid washing are two major means of controlling contaminants. However, antibiotic use could lead to increased antibiotic resistance, and the acid wash step stresses the fermenting yeast and has limited effectiveness. Bacteriophage endolysins such as LysA2 are lytic enzymes with the potential to contribute as antimicrobials to the fuel ethanol industries. Our goal was to evaluate the potential of yeast-derived LysA2 as a means of controlling Lactobacillaceae contamination. LysA2 intracellularly produced by Pichia pastoris showed activity comparable to Escherichia coli produced LysA2. Lactic Acid Bacteria (LAB) with the A4α peptidoglycan chemotype (L-Lys-D-Asp crosslinkage) were the most sensitive to LysA2, though a few from that chemotype were insensitive. Pichia -expressed LysA2, both secreted and intracellularly produced, successfully improved ethanol productivity and yields in glucose (YPD60) and sucrose-based (sugarcane juice) ethanol fermentations in the presence of a LysA2 susceptible LAB contaminant. LysA2 secreting Sacharomyces cerevisiae did not notably improve production in sugarcane juice, but it did control bacterial contamination during fermentation in YPD60. Secretion of LysA2 by the fermenting yeast, or adding it in purified form, are promising alternative tools to control LAB contamination during ethanol fermentation. Endolysins with much broader lytic spectrums than LysA2 could supplement or replace the currently used antibiotics or the acidic wash.

  20. Low toxicity of HfO2, SiO2, Al2O3 and CeO2 nanoparticles to the yeast, Saccharomyces cerevisiae.

    PubMed

    García-Saucedo, Citlali; Field, James A; Otero-Gonzalez, Lila; Sierra-Álvarez, Reyes

    2011-09-15

    Increasing use of nanomaterials necessitates an improved understanding of their potential impact on environment health. This study evaluated the cytotoxicity of nanosized HfO(2), SiO(2), Al(2)O(3) and CeO(2) towards the eukaryotic model organism Saccharomyces cerevisiae, and characterized their state of dispersion in bioassay medium. Nanotoxicity was assessed by monitoring oxygen consumption in batch cultures and by analysis of cell membrane integrity. CeO(2), Al(2)O(3), and HfO(2) nanoparticles were highly unstable in yeast medium and formed micron-sized, settleable agglomerates. A non-toxic polyacrylate dispersant (Dispex A40) was used to improve nanoparticle stability and determine the impact of enhanced dispersion on toxicity. None of the NPs tested without dispersant inhibited O(2) uptake by yeast at concentrations as high as 1000 mg/L. Dispersant supplementation only enhanced the toxicity of CeO(2) (47% at 1000 mg/L). Dispersed SiO(2) and Al(2)O(3) (1000 mg/L) caused cell membrane damage, whereas dispersed HfO(2) and CeO(2) did not cause significant disruption of membrane integrity at the same concentration. These results suggest that the O(2) uptake inhibition observed with dispersed CeO(2) NPs was not due to reduced cell viability. This is the first study evaluating toxicity of nanoscale HfO(2), SiO(2), Al(2)O(3) and CeO(2) to S. cerevisiae. Overall the results obtained demonstrate that these nanomaterials display low or no toxicity to yeast. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Yeast Based Sensors

    NASA Astrophysics Data System (ADS)

    Shimomura-Shimizu, Mifumi; Karube, Isao

    Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of yeast based sensors have been developed as analytical tools. Yeasts are known as facultative anaerobes. Facultative anaerobes can survive in both aerobic and anaerobic conditions. The yeast based sensor consisted of a DO electrode and an immobilized omnivorous yeast. In yeast based sensor development, many kinds of yeast have been employed by applying their characteristics to adapt to the analyte. For example, Trichosporon cutaneum was used to estimate organic pollution in industrial wastewater. Yeast based sensors are suitable for online control of biochemical processes and for environmental monitoring. In this review, principles and applications of yeast based sensors are summarized.

  2. Comparison of clinical and analytical performance of the Abbott Realtime High Risk HPV test to the performance of hybrid capture 2 in population-based cervical cancer screening.

    PubMed

    Poljak, Mario; Ostrbenk, Anja; Seme, Katja; Ucakar, Veronika; Hillemanns, Peter; Bokal, Eda Vrtacnik; Jancar, Nina; Klavs, Irena

    2011-05-01

    The clinical performance of the Abbott RealTime High Risk HPV (human papillomavirus) test (RealTime) and that of the Hybrid Capture 2 HPV DNA test (hc2) were prospectively compared in the population-based cervical cancer screening setting. In women >30 years old (n = 3,129), the clinical sensitivity of RealTime for detection of cervical intraepithelial neoplasia of grade 2 (CIN2) or worse (38 cases) and its clinical specificity for lesions of less than CIN2 (3,091 controls) were 100% and 93.3%, respectively, and those of hc2 were 97.4% and 91.8%, respectively. A noninferiority score test showed that the clinical specificity (P < 0.0001) and clinical sensitivity (P = 0.011) of RealTime were noninferior to those of hc2 at the recommended thresholds of 98% and 90%. In the total study population (women 20 to 64 years old; n = 4,432; 57 cases, 4,375 controls), the clinical sensitivity and specificity of RealTime were 98.2% and 89.5%, and those of hc2 were 94.7% and 87.7%, respectively. The analytical sensitivity and analytical specificity of RealTime in detecting targeted HPV types evaluated with the largest sample collection to date (4,479 samples) were 94.8% and 99.8%, and those of hc2 were 93.4% and 97.8%, respectively. Excellent analytical agreement between the two assays was obtained (kappa value, 0.84), while the analytical accuracy of RealTime was significantly higher than that of hc2. RealTime demonstrated high intralaboratory reproducibility and interlaboratory agreement with 500 samples retested 61 to 226 days after initial testing in two different laboratories. RealTime can be considered to be a reliable and robust HPV assay clinically comparable to hc2 for the detection of CIN2+ lesions in a population-based cervical cancer screening setting.

  3. Screen and effect analysis of immunostimulants for sea cucumber, Apostichopus japonicus

    NASA Astrophysics Data System (ADS)

    Li, Jiye; Sun, Xiuqin; Zheng, Fengrong; Hao, Linhua

    2009-02-01

    Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal’s coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides ( P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection ( P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection ( P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.

  4. Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

    PubMed Central

    Kouprina, Natalya; Kawamoto, Kensaku; Barrett, J. Carl; Larionov, Vladimir; Koi, Minoru

    1998-01-01

    In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ∼1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system. PMID:9647640

  5. Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

    PubMed

    Kobayashi, Takehiko; Sasaki, Mariko

    2017-01-01

    The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Electron transport chain in a thermotolerant yeast.

    PubMed

    Mejía-Barajas, Jorge A; Martínez-Mora, José A; Salgado-Garciglia, Rafael; Noriega-Cisneros, Ruth; Ortiz-Avila, Omar; Cortés-Rojo, Christian; Saavedra-Molina, Alfredo

    2017-04-01

    Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ m ), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca 2+ ions (Ca 2+ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30 o , while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.

  7. A novel method for purification of the endogenously expressed fission yeast Set2 complex.

    PubMed

    Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

    2014-05-01

    Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Zea mays Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

    PubMed Central

    Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity. PMID:22937104

  9. Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids.

    PubMed

    Hsu, Yu-Yi; Chou, Jui-Yu

    2017-01-01

    Mitochondria play a critical role in the generation of metabolic energy and are crucial for eukaryotic cell survival and proliferation. In most sexual eukaryotes, mitochondrial DNA (mtDNA) is inherited from only one parent in non-Mendelian inheritance in contrast to the inheritance of nuclear DNA. The model organism Saccharomyces cerevisiae is commonly used to study mitochondrial biology. It has two mating types: MATa and MATα. Previous studies have suggested that the mtDNA inheritance patterns in hybrid diploid cells depend on the genetic background of parental strains. However, the underlying mechanisms remain unclear. To elucidate the mechanisms, we examined the effects of environmental factors on the mtDNA inheritance patterns in hybrids obtained by crossing S. cerevisiae with its close relative S. paradoxus. The results demonstrated that environmental factors can influence mtDNA transmission in hybrid diploids, and that the inheritance patterns are strain dependent. The fitness competition assay results showed that the fitness differences can explain the mtDNA inheritance patterns under specific conditions. However, in this study, we found that fitness differences cannot fully be explained by mitochondrial activity in hybrids under stress conditions.

  10. Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids

    PubMed Central

    Hsu, Yu-Yi; Chou, Jui-Yu

    2017-01-01

    Mitochondria play a critical role in the generation of metabolic energy and are crucial for eukaryotic cell survival and proliferation. In most sexual eukaryotes, mitochondrial DNA (mtDNA) is inherited from only one parent in non-Mendelian inheritance in contrast to the inheritance of nuclear DNA. The model organism Saccharomyces cerevisiae is commonly used to study mitochondrial biology. It has two mating types: MATa and MATα. Previous studies have suggested that the mtDNA inheritance patterns in hybrid diploid cells depend on the genetic background of parental strains. However, the underlying mechanisms remain unclear. To elucidate the mechanisms, we examined the effects of environmental factors on the mtDNA inheritance patterns in hybrids obtained by crossing S. cerevisiae with its close relative S. paradoxus. The results demonstrated that environmental factors can influence mtDNA transmission in hybrid diploids, and that the inheritance patterns are strain dependent. The fitness competition assay results showed that the fitness differences can explain the mtDNA inheritance patterns under specific conditions. However, in this study, we found that fitness differences cannot fully be explained by mitochondrial activity in hybrids under stress conditions. PMID:28081193

  11. Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

    PubMed Central

    Kretschmer, M; Schellenberger, W; Otto, A; Kessler, R; Hofmann, E

    1987-01-01

    Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A. PMID:2825652

  12. Enhanced leavening ability of baker's yeast by overexpression of SNR84 with PGM2 deletion.

    PubMed

    Lin, Xue; Zhang, Cui-Ying; Bai, Xiao-Wen; Xiao, Dong-Guang

    2015-06-01

    Dough-leavening ability is one of the main aspects considered when selecting a baker's yeast strain for baking industry. Generally, modification of maltose metabolic pathway and known regulatory networks of maltose metabolism were used to increase maltose metabolism to improve leavening ability in lean dough. In this study, we focus on the effects of PGM2 (encoding for the phosphoglucomutase) and SNR84 (encoding for the H/ACA snoRNA) that are not directly related to both the maltose metabolic pathway and known regulatory networks of maltose metabolism on the leavening ability of baker's yeast in lean dough. The results show that the modifications on PGM2 and/or SNR84 are effective ways in improving leavening ability of baker's yeast in lean dough. Deletion of PGM2 decreased cellular glucose-1-phosphate and overexpression of SNR84 increased the maltose permease activity. These changes resulted in 11, 19 and 21% increases of the leavening ability for PGM2 deletion, SNR84 overexpression and SNR84 overexpression combining deleted PGM2, respectively.

  13. New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

    PubMed Central

    Cobo, Ana Cristina; Milán, Miguel; Al-Asmar, Nasser; García-Herrero, Sandra; Mir, Pere; Simón, Carlos

    2014-01-01

    The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation. PMID:24877108

  14. Yeast viability and concentration analysis using lens-free computational microscopy and machine learning

    NASA Astrophysics Data System (ADS)

    Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

    2017-03-01

    Research laboratories and the industry rely on yeast viability and concentration measurements to adjust fermentation parameters such as pH, temperature, and pressure. Beer-brewing processes as well as biofuel production can especially utilize a cost-effective and portable way of obtaining data on cell viability and concentration. However, current methods of analysis are relatively costly and tedious. Here, we demonstrate a rapid, portable, and cost-effective platform for imaging and measuring viability and concentration of yeast cells. Our platform features a lens-free microscope that weighs 70 g and has dimensions of 12 × 4 × 4 cm. A partially-coherent illumination source (a light-emitting-diode), a band-pass optical filter, and a multimode optical fiber are used to illuminate the sample. The yeast sample is directly placed on a complementary metal-oxide semiconductor (CMOS) image sensor chip, which captures an in-line hologram of the sample over a large field-of-view of >20 mm2. The hologram is transferred to a touch-screen interface, where a trained Support Vector Machine model classifies yeast cells stained with methylene blue as live or dead and measures cell viability as well as concentration. We tested the accuracy of our platform against manual counting of live and dead cells using fluorescent exclusion staining and a bench-top fluorescence microscope. Our regression analysis showed no significant difference between the two methods within a concentration range of 1.4 × 105 to 1.4 × 106 cells/mL. This compact and cost-effective yeast analysis platform will enable automatic quantification of yeast viability and concentration in field settings and resource-limited environments.

  15. The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

    PubMed

    Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

    2007-09-01

    Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

  16. Electro-optic guided-mode resonance tuning suppressible by optically induced screening in a vertically coupled hybrid GaN/Si microring resonator

    NASA Astrophysics Data System (ADS)

    Thubthimthong, B.; Sasaki, T.; Hane, K.

    2018-02-01

    GaN as a nanophotonic material has gained much attention in recent years. Using the hybrid GaN/Si platform, we report the electro-optic tuning of guided-mode resonance in a vertically coupled hybrid GaN/Si microring resonator operating in the 1.5 μm window with up to a 6 dB extinction ratio and a 1.5 MHz modulation frequency (test equipment limit). The electro-optic tuning could be optically suppressed by electron-hole-originated screening induced by an ultraviolet excitation at 325 nm. Our work may benefit in externally intervenable optical interconnects for uninterrupted secure photonic networks.

  17. Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

    PubMed

    Ward, Thomas A; Dudášová, Zuzana; Sarkar, Sovan; Bhide, Mangesh R; Vlasáková, Danuša; Chovanec, Miroslav; McHugh, Peter J

    2012-01-01

    Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

  18. Lysine methylation modulates the protein-protein interactions of yeast cytochrome C Cyc1p.

    PubMed

    Winter, Daniel L; Abeygunawardena, Dhanushi; Hart-Smith, Gene; Erce, Melissa A; Wilkins, Marc R

    2015-07-01

    In recent years, protein methylation has been established as a major intracellular PTM. It has also been proposed to modulate protein-protein interactions (PPIs) in the interactome. To investigate the effect of PTMs on PPIs, we recently developed the conditional two-hybrid (C2H) system. With this, we demonstrated that arginine methylation can modulate PPIs in the yeast interactome. Here, we used the C2H system to investigate the effect of lysine methylation. Specifically, we asked whether Ctm1p-mediated trimethylation of yeast cytochrome c Cyc1p, on lysine 78, modulates its interactions with Erv1p, Ccp1p, Cyc2p and Cyc3p. We show that the interactions between Cyc1p and Erv1p, and between Cyc1p and Cyc3p, are significantly increased upon trimethylation of lysine 78. This increase of interaction helps explain the reported facilitation of Cyc1p import into the mitochondrial intermembrane space upon methylation. This first application of the C2H system to the study of methyllysine-modulated interactions further confirms its robustness and flexibility. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Replication dynamics of the yeast genome.

    PubMed

    Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

    2001-10-05

    Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

  20. A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion

    PubMed Central

    Groux, Raphaël; Vincenzetti, Vincent

    2017-01-01

    In non-motile fungi, sexual reproduction relies on strong morphogenetic changes in response to pheromone signaling. We report here on a systematic screen for morphological abnormalities of the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions, which, upon visual screening, revealed a plethora of phenotypes affecting all stages of the mating process, including cell polarization, cell fusion and sporulation. Cell fusion relies on the formation of the fusion focus, an aster-like F-actin structure that is marked by strong local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondary screen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical for the coalescence of the fusion focus. Indeed, rng8Δ and rng9Δ mutant cells exhibit multiple stable dots at the cell-cell contact site, instead of the single focus observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, dependent on Myo51 and tropomyosin Cdc8. A tropomyosin mutant allele, which compromises Rng8/9 localization but not actin binding, similarly leads to multiple stable dots instead of a single focus. By contrast, myo51 deletion does not strongly affect fusion focus coalescence. We propose that focusing of the actin filaments in the fusion aster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments. PMID:28410370

  1. Antioxidant N-acetyltransferase Mpr1/2 of industrial baker's yeast enhances fermentation ability after air-drying stress in bread dough.

    PubMed

    Sasano, Yu; Takahashi, Shunsuke; Shima, Jun; Takagi, Hiroshi

    2010-03-31

    During bread-making processes, yeast cells are exposed to multiple stresses. Air-drying stress is one of the most harmful stresses by generation of reactive oxygen species (ROS). Previously, we discovered that the novel N-acetyltransferase Mpr1/2 confers oxidative stress tolerance by reducing intracellular ROS level in Saccharomyces cerevisiae Sigma1278b strain. In this study, we revealed that Japanese industrial baker's yeast possesses one MPR gene. The nucleotide sequence of the MPR gene in industrial baker's yeast was identical to the MPR2 gene in Sigma1278b strain. Gene disruption analysis showed that the MPR2 gene in industrial baker's yeast is involved in air-drying stress tolerance by reducing the intracellular oxidation levels. We also found that expression of the Lys63Arg and Phe65Leu variants with enhanced enzymatic activity and stability, respectively, increased the fermentation ability of bread dough after exposure to air-drying stress compared with the wild-type Mpr1. In addition, our recent study showed that industrial baker's yeast cells accumulating proline exhibited enhanced freeze tolerance in bread dough. Proline accumulation also enhanced the fermentation ability after air-drying stress treatment in industrial baker's yeast. Hence, the antioxidant enzyme Mpr1/2 could be promising for breeding novel yeast strains that are tolerant to air-drying stress. Copyright 2010 Elsevier B.V. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

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

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

  3. Yeast Infection (Vaginal)

    MedlinePlus

    Yeast infection (vaginal) Overview A vaginal yeast infection is a fungal infection that causes irritation, discharge and intense itchiness ... symptoms Causes The fungus candida causes a vaginal yeast infection. Your vagina naturally contains a balanced mix of yeast, including ...

  4. [Thermoresistance in Saccharomyces cerevisiae yeasts].

    PubMed

    Kaliuzhin, V A

    2011-01-01

    Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

  5. Candida species and other yeasts in the oral cavities of type 2 diabetic patients in Cali, Colombia

    PubMed Central

    Álvarez, María Inés; de Bernal, Matilde; Collazos, Andrés

    2013-01-01

    Objective: To determine the prevalence of Candida species and to study factors associated to oral cavity colonization in patients with type 2 diabetes mellitus. Methods: A total of 107 diabetics were classified into controlled and uncontrolled according to glycosylated hemoglobin values. Each patient was assessed for stimulated salivary flow rates, pH, and an oral rinse to search for yeast. The study also determined the state of oral health via Klein and Palmer CPO indexes for permanent dentition, dental plaque by O'Leary, and a periodontal chart. Results: We found yeasts in 74.8% of the patients. A total of 36 of the 52 subjects with controlled diabetes presented yeasts and 44 in the uncontrolled; no significant differences (p = 0.2) were noted among the presence of yeasts and the control of blood glucose. The largest number of isolates corresponded to C. albicans, followed by C. parapsilosis. Uncontrolled individuals presented a significantly higher percentage of yeast different from C. albicans (p = 0.049). Conclusions: We found a high percentage of Candida colonization and uncontrolled individuals had greater diversity of species. The wide range of CFU/mL found both in patients with oral candidiasis, as well as in those without it did not permit distinguishing between colonization and disease. We only found association between isolation of yeasts and the low rate of salivary flow. PMID:24892318

  6. Arsenic processing of yeast isolates IIB-As1 & IIB-As2 and production of glutathione under stress conditions.

    PubMed

    Muneer, Bushra; Lali, Tayyaba; Iqbal, Muhammad J; Shakoori, Farah R; Shakoori, Abdul R

    2016-10-01

    Four arsenic resistant yeast were isolated from the industrial wastewater. Two strains IIB-As1 and IIB-As2 identified as Candida tropicalis and Saccharomyces cerevisiae, respectively. IIB-As1 and IIB-As2 showed maximum arsenic resistance. IIB-As1 showed maximum growth at 35 °C whereas it was 30 °C for IIB-As2. The yeast isolate showed typical growth curves, but arsenic extended the lag phase. Glutathione plays an important role in metal tolerance. In the present study, As increased the level glutathione and non-protein thiols in yeast isolates. Removal of As from supernatant was analyzed using the atomic absorption spectrophotometer. They removed arsenic from the medium after 72 h of incubation. Both yeast strains efficiently removed arsenic from the industrial effluent when used individually or in consortia. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

    PubMed

    Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

    2014-09-01

    The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

    NASA Technical Reports Server (NTRS)

    Dalal, Sawan; Santa Maria, Sergio R.; Liddell, Lauren; Bhattacharya, Sharmila

    2017-01-01

    BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful

  9. Final screening round of the NELSON lung cancer screening trial: the effect of a 2.5-year screening interval.

    PubMed

    Yousaf-Khan, Uraujh; van der Aalst, Carlijn; de Jong, Pim A; Heuvelmans, Marjolein; Scholten, Ernst; Lammers, Jan-Willem; van Ooijen, Peter; Nackaerts, Kristiaan; Weenink, Carla; Groen, Harry; Vliegenthart, Rozemarijn; Ten Haaf, Kevin; Oudkerk, Matthijs; de Koning, Harry

    2017-01-01

    In the USA annual lung cancer screening is recommended. However, the optimal screening strategy (eg, screening interval, screening rounds) is unknown. This study provides results of the fourth screening round after a 2.5-year interval in the Dutch-Belgian Lung Cancer Screening trial (NELSON). Europe's largest, sufficiently powered randomised lung cancer screening trial was designed to determine whether low-dose CT screening reduces lung cancer mortality by ≥25% compared with no screening after 10 years of follow-up. The screening arm (n=7915) received screening at baseline, after 1 year, 2 years and 2.5 years. Performance of the NELSON screening strategy in the final fourth round was evaluated. Comparisons were made between lung cancers detected in the first three rounds, in the final round and during the 2.5-year interval. In round 4, 46 cancers were screen-detected and there were 28 interval cancers between the third and fourth screenings. Compared with the second round screening (1-year interval), in round 4 a higher proportion of stage IIIb/IV cancers (17.3% vs 6.8%, p=0.02) and higher proportions of squamous-cell, bronchoalveolar and small-cell carcinomas (p=0.001) were detected. Compared with a 2-year interval, the 2.5-year interval showed a higher non-significant stage distribution (stage IIIb/IV 17.3% vs 5.2%, p=0.10). Additionally, more interval cancers manifested in the 2.5-year interval than in the intervals of previous rounds (28 vs 5 and 28 vs 19). A 2.5-year interval reduced the effect of screening: the interval cancer rate was higher compared with the 1-year and 2-year intervals, and proportion of advanced disease stage in the final round was higher compared with the previous rounds. ISRCTN63545820. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. High-throughput analysis of yeast replicative aging using a microfluidic system

    PubMed Central

    Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

    2015-01-01

    Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

  11. Protocols and programs for high-throughput growth and aging phenotyping in yeast.

    PubMed

    Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

    2015-01-01

    In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

  12. Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans.

    PubMed

    Yun, Dae Gyu; Lee, Dong Gun

    2016-11-01

    Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100μM silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K + and accumulation of cytoplasmic and mitochondrial Ca 2+ . Among these phenomena, mitochondrial Ca 2+ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca 2+ in apoptosis by employing 20mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca 2+ signaling in C. albicans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Reconstruction of the Evolutionary History of Saccharomyces cerevisiae x S. kudriavzevii Hybrids Based on Multilocus Sequence Analysis

    PubMed Central

    Peris, David; Lopes, Christian A.; Arias, Armando; Barrio, Eladio

    2012-01-01

    In recent years, interspecific hybridization and introgression are increasingly recognized as significant events in the evolution of Saccharomyces yeasts. These mechanisms have probably been involved in the origin of novel yeast genotypes and phenotypes, which in due course were to colonize and predominate in the new fermentative environments created by human manipulation. The particular conditions in which hybrids arose are still unknown, as well as the number of possible hybridization events that generated the whole set of natural hybrids described in the literature during recent years. In this study, we could infer at least six different hybridization events that originated a set of 26 S. cerevisiae x S. kudriavzevii hybrids isolated from both fermentative and non-fermentative environments. Different wine S. cerevisiae strains and European S. kudriavzevii strains were probably involved in the hybridization events according to gene sequence information, as well as from previous data on their genome composition and ploidy. Finally, we postulate that these hybrids may have originated after the introduction of vine growing and winemaking practices by the Romans to the present Northern vine-growing limits and spread during the expansion of improved viticulture and enology practices that occurred during the Late Middle Ages. PMID:23049811

  14. Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity.

    PubMed

    Yang, Yijin; Xia, Yongjun; Lin, Xiangna; Wang, Guangqiang; Zhang, Hui; Xiong, Zhiqiang; Yu, Haiyan; Yu, Jianshen; Ai, Lianzhong

    2018-06-01

    Producing alcoholic beverages with novel flavor are desirable for winemakers. We created fermenting yeast with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Strategies of ethanol domestication, ultraviolet mutagenesis (UV) and protoplast fusion were conducted to create yeast hybrids with excellent oenological characteristic. The obtained diploid hybrid F23 showed a cell viability of 6.2% under 25% ethanol, whereas its diploid parental strains could not survive under 20% ethanol. During Chinese rice wine-making, compared to diploid parents, F23 produced 7.07%-12.44% higher yield of ethanol. Flavor analysis indicated that the total content of flavor compounds in F23 wine was 19.99-26.55% higher than that of parent wines. Specifically, F23 exhibited higher capacity in producing 2-phenylethanol, short-chain and long-chain fatty-acid ethyl-ester than diploid parents. Compared to diploid parents, F23 introduced more flavor contributors with odor activity values (OAVs) above one to Chinese rice wine, and those contributors were found with higher OAVs. Based on principal component analysis (PCA), the flavor characteristic of F23 wine was similar to each of parent wine. Additionally, sensory evaluation showed that F23 wine was highly assessed for its intensive levels in fruit-aroma, alcohol-aroma and mouthfeel. Hybrid F23 not only displayed superior flavor production and oenological performance in making Chinese rice wine, but also could act as potential "mixed-like" starter to enrich wine style and differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy.

    PubMed

    Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

    2017-01-01

    Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, Sc Alt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1 , alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display Lk Alt1 and Kl Alt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that Kl Alt1 and Lk Alt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that Sc Alt2 conserves 64% identity with

  16. Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

    PubMed Central

    Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

    2017-01-01

    Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64% identity with LkAlt1

  17. Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process.

    PubMed

    de Melo Pereira, Gilberto Vinícius; Soccol, Vanete Thomaz; Pandey, Ashok; Medeiros, Adriane Bianchi Pedroni; Andrade Lara, João Marcos Rodrigues; Gollo, André Luiz; Soccol, Carlos Ricardo

    2014-10-01

    During wet processing of coffee, the ripe cherries are pulped, then fermented and dried. This study reports an experimental approach for target identification and selection of indigenous coffee yeasts and their potential use as starter cultures during the fermentation step of wet processing. A total of 144 yeast isolates originating from spontaneously fermenting coffee beans were identified by molecular approaches and screened for their capacity to grow under coffee-associated stress conditions. According to ITS-rRNA gene sequencing, Pichia fermentans and Pichia kluyveri were the most frequent isolates, followed by Candida Candida glabrata, quercitrusa, Saccharomyces sp., Pichia guilliermondii, Pichia caribbica and Hanseniaspora opuntiae. Nine stress-tolerant yeast strains were evaluated for their ability to produce aromatic compounds in a coffee pulp simulation medium and for their pectinolytic activity. P. fermentans YC5.2 produced the highest concentrations of flavor-active ester compounds (viz., ethyl acetate and isoamyl acetate), while Saccharomyces sp. YC9.15 was the best pectinase-producing strain. The potential impact of these selected yeast strains to promote flavor development in coffee beverages was investigated for inoculating coffee beans during wet fermentation trials at laboratory scale. Inoculation of a single culture of P. fermentans YC5.2 and co-culture of P. fermentans YC5.2 and Saccharomyces sp. YC9.15 enhanced significantly the formation of volatile aroma compounds during the fermentation process compared to un-inoculated control. The sensory analysis indicated that the flavor of coffee beverages was influenced by the starter cultures, being rated as having the higher sensory scores for fruity, buttery and fermented aroma. This demonstrates a complementary role of yeasts associated with coffee quality through the synthesis of yeast-specific volatile constituents. The yeast strains P. fermentans YC5.2 and Saccharomyces sp. YC9.15 have a great

  18. Effect of Spin-Orbit Coupling on the Actinide Dioxides AnO2 (An=Th, Pa, U, Np, Pu, and Am): A Screened Hybrid Density Functional Study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wen, Xiaodong; Martin, Richard L.; Roy, Lindsay E.

    2012-10-21

    We present a systematic comparison of the lattice structures, electronic density of states, and band gaps of actinide dioxides, AnO₂ (An=Th, Pa, U, Np, Pu, and Am) predicted by the Heyd-Scuseria-Ernzerhof screened hybrid density functional (HSE) with the self-consistent inclusion of spin-orbit coupling(SOC). The computed HSE lattice constants and band gaps of AnO₂ are in consistently good agreement with the available experimental data across the series, and differ little from earlier HSE results without SOC. ThO₂ is a simple band insulator (f⁰), while PaO₂, UO₂, and NpO₂ are predicted to be Mott insulators. The remainders (PuO₂ and AmO₂) show considerablemore » O2p/An5f mixing and are classified as charge-transfer insulators. We also compare our results for UO₂, NpO₂, and PuO₂with the PBE+U, self interaction correction (SIC), and dynamic mean-field theory (DMFT) many-body approximations.« less

  19. Stay-green ranking and maturity of corn hybrids: 2. Effects on the performance of lactating dairy cows.

    PubMed

    Arriola, K G; Kim, S C; Staples, C R; Adesogan, A T

    2012-02-01

    To address producer concerns that feeding high stay-green (SG) corn hybrids is associated with decreased performance and health problems in dairy cows, this study examined how the performance of cows was affected by feeding hybrids with contrasting SG rankings and maturities. Two near-isogenic corn hybrids with high (HSG; Croplan Genetics 691, Croplan Genetics, St. Paul, MN) and low (LSG; Croplan Genetics 737) SG rankings were grown on separate halves of a 10-ha field, harvested at 27% (maturity 1) or 35% (maturity 2) dry matter (DM) and ensiled in bag silos for 84 and 77 d, respectively. A further treatment involved addition of water (15 L/t) to the HSG maturity 1 hybrid during packing to compound the potential negative effects of excess water in the HSG hybrid. Each of the resulting silages was included in a total mixed ration consisting of 35, 55, and 10% (DM basis) of corn silage, concentrate, and alfalfa hay, respectively. In experiment 1, the total mixed ration was fed for ad libitum consumption twice daily to 30 Holstein cows (92±18 d in milk). This experiment had a completely randomized design and consisted of two 28-d periods, each with 14 d for adaptation and 14 d for sample collection. In experiment 2, the ruminal fermentation of the diets was measured using 5 ruminally cannulated cows on the last day of three 15-d periods. Ruminal contraction rate (2.28±0.14 contractions/min), milk yield (36.7±1.3 kg/d), yield of milk protein (1.1±0.03 kg/d), and concentration of milk protein (2.9±0.03%) were not affected by treatment. Feeding diets containing HSG instead of LSG reduced intake of crude protein (CP) and neutral detergent fiber, digestibility of neutral detergent fiber, and concentrations of ruminal total volatile fatty acids (VFA) and milk fat when the hybrids were harvested at 27% DM but not 35% DM. Across maturity stages, feeding diets containing HSG instead of LSG decreased DM and CP digestibility, increased rectal temperature and plasma

  20. Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells

    PubMed Central

    Koyama, Sumihiro; Tsubouchi, Taishi; Usui, Keiko; Uematsu, Katsuyuki; Tame, Akihiro; Nogi, Yuichi; Ohta, Yukari; Hatada, Yuji; Kato, Chiaki; Miwa, Tetsuya; Toyofuku, Takashi; Nagahama, Takehiko; Konishi, Masaaki; Nagano, Yuriko; Abe, Fumiyoshi

    2015-01-01

    The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications. PMID:26187908

  1. Rapid screening and identification of multi-class substances of very high concern in textiles using liquid chromatography-hybrid linear ion trap orbitrap mass spectrometry.

    PubMed

    Zhang, Li; Luo, Xin; Niu, Zengyuan; Ye, Xiwen; Tang, Zhixu; Yao, Peng

    2015-03-20

    A new analytical method was established and validated for the analysis of 19 substances of very high concern (SVHCs) in textiles, including phthalic acid esters (PAEs), organotins (OTs), perfluorochemicals (PFCs) and flame retardants (FRs). After ultrasonic extraction in methanol, the textile samples were analyzed by high performance liquid chromatography-hybrid linear ion trap Orbitrap high resolution mass spectrometry (HPLC-LTQ/Orbitrap). The values of LOQ were in the range of 2-200mg/kg. Recoveries at two levels (at the LOQ and at half the limit of regulation) ranged from 68% to 120%, and the repeatability was lower than 13%. This method was successfully applied to the screening of SVHCs in commercial textile samples and is useful for the fast screening of various SVHCs. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family

    PubMed Central

    Sun, Qi; Manns, David C.; Sacks, Gavin L.; Mansfield, Anna Katharine; Luby, James J.; Londo, Jason P.; Reisch, Bruce I.; Cadle-Davidson, Lance E.; Fennell, Anne Y.

    2016-01-01

    In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species. PMID:26974672

  3. Caspase-2 associates with FAN through direct interaction and overlapping functionality.

    PubMed

    Forsberg, Jeremy; Li, Xinge; Zamaraev, Aleksey V; Panaretakis, Theocharis; Zhivotovsky, Boris; Olsson, Magnus

    2018-05-23

    Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN. Lipidomics excluded the involvement of caspase-2 in the generation of ceramide species, but caspase-2-dependent deregulation of IL-6 release, vesicular size and delayed cell relocation supported an association between caspase-2 and FAN. Collectively, these data identify a novel caspase-2-interacting factor, FAN, and expand the role for the enzyme in seemingly non-apoptotic cellular mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. An Integrated In Silico Method to Discover Novel Rock1 Inhibitors: Multi- Complex-Based Pharmacophore, Molecular Dynamics Simulation and Hybrid Protocol Virtual Screening.

    PubMed

    Chen, Haining; Li, Sijia; Hu, Yajiao; Chen, Guo; Jiang, Qinglin; Tong, Rongsheng; Zang, Zhihe; Cai, Lulu

    2016-01-01

    Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) is an important regulator of focal adhesion, actomyosin contraction and cell motility. In this manuscript, a combination of the multi-complex-based pharmacophore (MCBP), molecular dynamics simulation and a hybrid protocol of a virtual screening method, comprised of multipharmacophore- based virtual screening (PBVS) and ensemble docking-based virtual screening (DBVS) methods were used for retrieving novel ROCK1 inhibitors from the natural products database embedded in the ZINC database. Ten hit compounds were selected from the hit compounds, and five compounds were tested experimentally. Thus, these results may provide valuable information for further discovery of more novel ROCK1 inhibitors.

  5. 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

  6. Mitochondrial introgression suggests extensive ancestral hybridization events among Saccharomyces species.

    PubMed

    Peris, David; Arias, Armando; Orlić, Sandi; Belloch, Carmela; Pérez-Través, Laura; Querol, Amparo; Barrio, Eladio

    2017-03-01

    Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several worldwide distributed wild Saccharomyces species and their hybrids isolated from different sources and geographic origins. We demonstrated the existence of several recombination points in mitochondrial region COX2-ORF1, likely mediated by either the activity of the protein encoded by the ORF1 (F-SceIII) gene, a free-standing homing endonuclease, or mostly facilitated by A+T tandem repeats and regions of integration of GC clusters. These introgressions were shown to occur among strains of the same species and among strains of different species, which suggests a complex model of Saccharomyces evolution that involves several ancestral hybridization events in wild environments. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Non-interferometric quantitative phase imaging of yeast cells

    NASA Astrophysics Data System (ADS)

    Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

  8. Clinical utility of a DNA probe to 17p11.2 in screening of patients with a peripheral neuropathy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blancato, J.; Precht, K.; Meck, J.

    1994-09-01

    We assessed the usefulness of in situ hybridization with a DNA probe to the area of chromosome 17 at p11.2 as a diagnostic tool for screening for Charcot Marte Tooth 1A (CMT 1A). In situ hybridization with a probe to 17p11.2 was performed on fixed lymphocytes from the following groups of individuals: (1) normal controls; (2) patients evoking a strong clinical suspicion of CMT 1A; and (3) 3 families with an apparent autosomal dominant peripheral neuropathy of unknown diagnoses. Group 2 patients had evidence of demyelination as defined by nerve conduction of less that 50% of the normal mean ormore » terminal latency greater than 50% of the normal mean in conduction studies. Analysis of interphase cells hybridized with a cosmid DNA probe to 17p11.2 requires inclusion of a normal control with each trial and masked observer. Due to the size of the target DNA and the nature of the centromeric heterochromatin, the scoring of this probe is more subjective than centromere probes. For example, if the two 17 chromosomes are decondensed as in interphase, two tandem signals may be visualized as one. Results from duplication positive patients demonstrate a large proportion of cells with two closely aligned, but separate, signals with an additional single signal. Normal results demonstrate a majority of cells with two separate signals representing both normal homologues. None of the 3 families with questionable diagnosis revealed a duplication at the region, reinforcing our belief that a clinical diagnosis is the most discriminating tool available for diagnosis of CMT 1A. We concur with Boylan that molecular analysis for CMT 1A is useful for establishing a diagnosis of CMT 1A, but is not a primary differential diagnostic test. The yield in screening patients without physiologic evidence of demyelination is likely to be low. We further find that the use of in situ hybridization is a simple method of performing the duplication analysis.« less

  9. Sensitivity of fructose-1,6-biphosphatase from yeast, liver and skeletal muscle to fructose-2,6-biphosphate and 5'-adenosine monophosphate.

    PubMed

    von Herrath, M; Holzer, H

    1988-05-01

    As a prerequisite for future studies on the possible effect of sulphite, an anti-microbial agent, on gluconeogenesis in yeast, a comparative study of fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis, from yeast, liver and skeletal muscle is reported. In contrast to FBPase from yeast or liver, FBPase from skeletal muscle is approximately 1000-fold more sensitive to inhibition by 5' adenosine monophosphate and 30 to 250-fold less sensitive to inhibition by fructose-2,6-bisphosphate. The kinetic properties of the FBPases, determined by the ratios R(Mg2+/Mn2+) and R (pH 7/9) of the enzyme activities, measured at 10 mM Mg2+ and 2 mM Mn2+ and at pH 7.0 and 9.0, respectively, show a drastic difference between the skeletal muscle and the yeast or liver enzymes. The data support the idea that the enzymes from yeast and liver function in gluconeogenesis, whereas the enzyme from skeletal muscle is involved in other biological functions.

  10. High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

    PubMed

    Kassir, Yona

    2017-01-01

    Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

  11. Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-d-Glucose

    PubMed Central

    Rincón, Ana M.; Codón, Antonio C.; Castrejón, Francisco; Benítez, Tahía

    2001-01-01

    We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-d-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-d-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties. PMID:11526034

  12. Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes.

    PubMed

    Leal, Gildemberg Amorim; Gomes, Luiz Humberto; Efraim, Priscilla; de Almeida Tavares, Flavio Cesar; Figueira, Antonio

    2008-08-01

    Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

  13. Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

    PubMed Central

    Lukša, Juliana; Podoliankaitė, Monika; Vepštaitė, Iglė; Strazdaitė-Žielienė, Živilė; Urbonavičius, Jaunius

    2015-01-01

    Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property. PMID:25710965

  14. Comparison of carbohydrate sources in yeast-fermentation CO2 generators for mosquito surveillance

    USDA-ARS?s Scientific Manuscript database

    Mosquito surveillance in remote areas with limited access to canisters of CO2 or dry ice will benefit from an effective alternative CO2 source, such as the natural production of CO2 from yeast fermentation of several carbohydrate sources. In this study, we document the differences in mosquito and n...

  15. Different selective pressures lead to different genomic outcomes as newly-formed hybrid yeasts evolve.

    PubMed

    Piotrowski, Jeff S; Nagarajan, Saisubramanian; Kroll, Evgueny; Stanbery, Alison; Chiotti, Kami E; Kruckeberg, Arthur L; Dunn, Barbara; Sherlock, Gavin; Rosenzweig, Frank

    2012-04-02

    Interspecific hybridization occurs in every eukaryotic kingdom. While hybrid progeny are frequently at a selective disadvantage, in some instances their increased genome size and complexity may result in greater stress resistance than their ancestors, which can be adaptively advantageous at the edges of their ancestors' ranges. While this phenomenon has been repeatedly documented in the field, the response of hybrid populations to long-term selection has not often been explored in the lab. To fill this knowledge gap we crossed the two most distantly related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum, and established a mixed population of homoploid and aneuploid hybrids to study how different types of selection impact hybrid genome structure. As temperature was raised incrementally from 31°C to 46.5°C over 500 generations of continuous culture, selection favored loss of the S. uvarum genome, although the kinetics of genome loss differed among independent replicates. Temperature-selected isolates exhibited greater inherent and induced thermal tolerance than parental species and founding hybrids, and also exhibited ethanol resistance. In contrast, as exogenous ethanol was increased from 0% to 14% over 500 generations of continuous culture, selection favored euploid S. cerevisiae x S. uvarum hybrids. Ethanol-selected isolates were more ethanol tolerant than S. uvarum and one of the founding hybrids, but did not exhibit resistance to temperature stress. Relative to parental and founding hybrids, temperature-selected strains showed heritable differences in cell wall structure in the forms of increased resistance to zymolyase digestion and Micafungin, which targets cell wall biosynthesis. This is the first study to show experimentally that the genomic fate of newly-formed interspecific hybrids depends on the type of selection they encounter during the course of evolution, underscoring the importance of the ecological theatre in

  16. Field screening of experimental corn hybrids and inbred lines for multiple ear-feeding insect resistance.

    PubMed

    Ni, Xinzhi; Xu, Wenwei; Krakowsky, Matthew D; Buntin, G David; Brown, Steve L; Lee, R Dewey; Coy, Anton E

    2007-10-01

    Identifying and using native insect resistance genes is the core of integrated pest management. In this study, 10 experimental corn, Zea mays L., hybrids and 10 inbred lines were screened for resistance to major ear-feeding insects in the southeastern Coastal Plain region of the United States during 2004 and 2005. Ear-feeding insect damage was assessed at harvest by visual damage rating for the corn earworm, Helicoverpa zea (Boddie), and by the percentage of kernels damaged by the maize weevil, Sitophilus zeamais Motschulsky, and stink bugs [combination of Euschistus servus (Say) and southern green stink bug, Nezara viridula (L.)]. Among the eight inbred lines and two control populations examined, C3S1B73-5b was resistant to corn earworm, maize weevil, and stink bugs. In contrast, C3S1B73-4 was resistant to corn earworm and stink bugs, but not to maize weevil. In a similar manner, the corn hybrid S1W*CML343 was resistant to all three ear-feeding insects, whereas hybrid C3S1B73-3*Tx205 was resistant to corn earworm and maize weevil in both growing seasons, but susceptible to stink bugs in 2005. The silk-feeding bioassay showed that corn earworm developed better on corn silk than did fall armyworm. Among all phenotypic traits examined (i.e., corn ear size, husk extension, and husk tightness), only corn ear size was negatively correlated to corn earworm damage in the inbred lines examined, whereas only husk extension (i.e., coverage) was negatively correlated to both corn earworm and maize weevil damage on the experimental hybrids examined. Such information could be used to establish a baseline for developing agronomically elite corn germplasm that confers multiple ear-feeding insect resistance.

  17. Arabidopsis mitochondrial voltage-dependent anion channel 3 (AtVDAC3) protein interacts with thioredoxin m2.

    PubMed

    Zhang, Min; Takano, Tetsuo; Liu, Shenkui; Zhang, Xinxin

    2015-05-08

    Voltage-dependent anion channels (VDACs) are conserved mitochondrial outer membrane proteins. A yeast two-hybrid screen identified interaction between Arabidopsis VDAC3 and the chloroplast protein thioredoxin m2 (AtTrx m2). This was confirmed via pull-down assay. A bimolecular fluorescence complementation assay located the interaction in mitochondria. AtVDAC3 and AtTrx m2 transcripts were expressed in multiple tissues and up-regulated by abiotic stress. Under NaCl stress, AtVDAC3 overexpression inhibited growth and increased H2O2 accumulation, while AtTrx m2 overexpression conferred resistance to NaCl and reduced H2O2. Results indicate that both AtVDAC3 and AtTrx m2 are involved in ROS signaling and play opposite roles in NaCl stress response. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Exploring the Yeast Acetylome Using Functional Genomics

    PubMed Central

    Duffy, Supipi Kaluarachchi; Friesen, Helena; Baryshnikova, Anastasia; Lambert, Jean-Philippe; Chong, Yolanda T.; Figeys, Daniel; Andrews, Brenda

    2014-01-01

    SUMMARY Lysine acetylation is a dynamic posttranslational modification with a well-defined role in regulating histones. The impact of acetylation on other cellular functions remains relatively uncharacterized. We explored the budding yeast acetylome with a functional genomics approach, assessing the effects of gene overexpression in the absence of lysine deacetylases (KDACs). We generated a network of 463 synthetic dosage lethal (SDL) interactions involving class I and II KDACs, revealing many cellular pathways regulated by different KDACs. A biochemical survey of genes interacting with the KDAC RPD3 identified 72 proteins acetylated in vivo. In-depth analysis of one of these proteins, Swi4, revealed a role for acetylation in G1-specific gene expression. Acetylation of Swi4 regulates interaction with its partner Swi6, both components of the SBF transcription factor. This study expands our view of the yeast acetylome, demonstrates the utility of functional genomic screens for exploring enzymatic pathways, and provides functional information that can be mined for future studies. PMID:22579291

  19. Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

    PubMed

    Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

    2012-01-01

    In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

  20. Study of amyloids using yeast

    PubMed Central

    Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

    2012-01-01

    Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100

  1. A ribosome-dependent GTPase from yeast distinct from elongation factor 2.

    PubMed Central

    Skogerson, L; Wakatama, E

    1976-01-01

    Three proteins required for poly(U)-directed polyphenylalanine synthesis have been separated from yeast. Two of the factors correspond to the elongation factors 1 and 2 described for other eukaryotic systems, according to the criteria of phenylalanyl-tRNA binding and diphtheria toxin-catalyzed ADP-ribosylation. The third protein, while absolutely required for polyphenylalanine synthesis, was a more active ribosome-dependent GTPase than elongation factor 2. PMID:174100

  2. Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

    PubMed Central

    Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Dubiez, Etienne; Mechulam, Yves; Schmitt, Emmanuelle

    2013-01-01

    Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex. PMID:23193270

  3. The yeast genome may harbor hypoxia response elements (HRE).

    PubMed

    Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

    2007-01-01

    The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

  4. Bioflavoring by non-conventional yeasts in sequential beer fermentations.

    PubMed

    Holt, Sylvester; Mukherjee, Vaskar; Lievens, Bart; Verstrepen, Kevin J; Thevelein, Johan M

    2018-06-01

    Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

    PubMed

    Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

    2017-03-31

    The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Metabolite profiling of microfluidic cell culture conditions for droplet based screening.

    PubMed

    Bjork, Sara M; Sjostrom, Staffan L; Andersson-Svahn, Helene; Joensson, Haakan N

    2015-07-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.

  7. Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

    PubMed Central

    Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

    2012-01-01

    Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

  8. 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

  9. Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Hiraki, Toshiki; Usui, Keiko; Abe, Fumiyoshi

    2010-12-01

    Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp-) strains under pressures of 15-25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp- cells to grow at 15-25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

  10. Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

    PubMed

    Chen, Da-Chung; Chen, Li-Yu; Ling, Qing-Dong; Wu, Meng-Hsueh; Wang, Ching-Tang; Suresh Kumar, S; Chang, Yung; Munusamy, Murugan A; Alarfajj, Abdullah A; Wang, Han-Chow; Hsu, Shih-Tien; Higuchi, Akon

    2014-05-01

    The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Triacetic acid lactone production in industrial Saccharomyces yeast strains

    USDA-ARS?s Scientific Manuscript database

    Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into thirteen industrial yeast strains of varied genetic background. TAL produ...

  12. Mutation Screening of Her-2, N-ras and Nf1 Genes in Brain Tumor Biopsies.

    PubMed

    Yapijakis, Christos; Adamopoulou, Maria; Tasiouka, Konstantina; Voumvourakis, Costas; Stranjalis, George

    2016-09-01

    A deeper understanding of the complex molecular pathology of brain malignancies is needed in order to develop more effective and targeted therapies of these highly lethal disorders. In an effort to further enlighten the molecular pathology of brain oncogenesis involving the her-2 (erbB-2/neu/ngl)/N-ras/nf1 pathway, we screened the genotypes of specimens from various types of brain tumors. The studied specimens included 35 biopsies of four general categories: 13 neuroglial tumors (4 astrocytomas, 2 oligodendrogliomas, 7 glioblastomas multiforme), 14 meningiomas, 3 other nervous system tumors (2 schwannomas, 1 craniopharyngioma) and 5 metastatic tumors (such as lung carcinomas and chronic myelocytic leukemia). Screening for most common mutations in oncogenes her-2, N-ras and tumor suppressor gene nf1 was conducted with molecular hybridization techniques (Southern blotting, dot blot and single-strand conformational polymorphism (SSCP) analysis, respectively), and was confirmed by DNA sequencing. Gene amplification of her-2 was observed in only two cases (6%), namely in one glioblastoma and in one meningioma. Screening of 3 hot spot codons of the N-ras gene (12, 13 and 61) and subsequent DNA sequencing revealed mutations in 19 biopsies encompassing all categories (54%). Screening for mutations in exons of the nf1 gene by SSCP analysis detected a novel nonsense mutation in exon 31 in a unique case of a glioblastoma biopsy (3%) taken from a patient without neurofibromatosis type I. Activated N-ras appears to be a major oncogene in brain oncogenesis, exhibiting the most important role in the her-2/N-ras/nf1 pathway. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  14. Oleaginous yeasts from Antarctica: Screening and preliminary approach on lipid accumulation.

    PubMed

    Viñarta, Silvana C; Angelicola, M Virginia; Barros, J Maximiliano; Fernández, Pablo M; Mac Cormak, Walter; Aybar, Manuel J; de Figueroa, Lucía I C

    2016-12-01

    The capability of 17 Rhodotorula spp. isolated from Antarctica to accumulate intracellular lipids in nitrogen-limited medium was investigated. As results, 10 isolates were selected by Nile red staining, while 12 isolates were selected as oleaginous by analysis of total lipid content (20.4-73%, w/w of dry biomass). The higher lipid production and accumulation was exhibited for six strains belonging to three species of Rhodotorula (Rhodotorula glutinis, Rhodotorula glacialis, and Rhodotorula laryngis). This is the first report where R. laryngis have been identified within oleaginous specie. Lipid accumulation was evaluated comparatively in two nitrogen-limited glucose-based media (MI and MII). MI (low C/N ratio) was more suitable for biomass and lipid production while in MII (high C/N ratio) total lipid content was improved. R. glutinis R4, R. glacialis R15, and R. glutinis R48 showed high lipid concentrations (4.65-6.93 g L -1 ) and they were able to accumulate large amounts of lipids per gram of biomass (47-77%, w/w). A similar profile in fatty acids composition and content of neutral lipids to vegetable oils was observed, indicating that lipids produced by oleaginous Antarctic yeasts can be considered an alternative feedstock for biodiesel production. Antarctica represents an important source of oleaginous yeasts with adaptive capabilities to accumulate considerable amounts of lipids with biotechnological interest at 15 °C and 25 °C. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Yeast β-1,6-glucan is a primary target for the Saccharomyces cerevisiae K2 toxin.

    PubMed

    Lukša, Juliana; Podoliankaitė, Monika; Vepštaitė, Iglė; Strazdaitė-Žielienė, Živilė; Urbonavičius, Jaunius; Servienė, Elena

    2015-04-01

    Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered.

    PubMed

    Jolly, Neil P; Varela, Cristian; Pretorius, Isak S

    2014-03-01

    Saccharomyces cerevisiae and grape juice are 'natural companions' and make a happy wine marriage. However, this relationship can be enriched by allowing 'wild' non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to 'the next level' if there are no spoilage yeast present and if the 'wine yeast' - S. cerevisiae - is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various 'matchmaking' strategies (e.g. cellar hygiene, pH, SO2 , temperature and nutrient management) to keep 'spoilers' (e.g. Dekkera bruxellensis) at bay, and allow 'compatible' wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a 'two is company, three is a crowd' scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present 'single-species' and 'multi-species' ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called 'natural' wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Antimicrobial activity of yeasts against some pathogenic bacteria

    PubMed Central

    Younis, Gamal; Awad, Amal; Dawod, Rehab E.; Yousef, Nehal E.

    2017-01-01

    Aim: This study was designed to isolate and identify yeast species from milk and meat products, and to test their antimicrobial activity against some bacterial species. Materials and Methods: A total of 160 milk and meat products samples were collected from random sellers and super markets in New Damietta city, Damietta, Egypt. Samples were subjected to yeast isolation procedures and tested for its antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. In addition, all yeast species isolates were subjected to polymerase chain reaction (PCR) for detection of khs (kievitone hydratase) and pelA (pectate degrading enzyme)genes. Results: The recovery rate of yeasts from sausage was 20% (2/10) followed by kareish cheese, processed cheese, and butter 10% (1/10) each as well as raw milk 9% (9/100), and fruit yoghurt 30% (6/20). Different yeast species were recovered, namely, Candida kefyr (5 isolates), Saccharomyces cerevisiae (4 isolates), Candida intermedia (3 isolates), Candida tropicalis (2 isolates), Candida lusitaniae (2 isolates), and Candida krusei (1 isolate). khs gene was detected in all S. cerevisiae isolates, however, pelA gene was not detected in all identified yeast species. Antimicrobial activity of recovered yeasts against the selected bacterial species showed high activity with C. intermedia against S. aureus and E. coli, C. kefyr against E. coli, and C. lusitaniae against S. aureus. Moderate activities were obtained with C. tropicalis, C. lusitaniae, and S. cerevisiae against E. coli; meanwhile, all the tested yeasts revealed a very low antimicrobial activity against P. aeruginosa. Conclusion: The obtained results confirmed that some kinds of yeasts have the ability to produce antimicrobial compounds that could inhibit some pathogenic and spoilage bacteria and these antimicrobial activity of yeasts enables them to be one of the novel agents in controlling spoilage of food. PMID:28919693

  18. Heavy metal removal by caustic-treated yeast immobilized in alginate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, Y.; Wilkins, E.

    1995-12-31

    Saccharomyces cerevisiae yeast biomass was treated with hot alkali to increase its biosorption capacity for heavy metals and then was immobilized in alginate gel. Biosorption capacities for Cu{sup 2+}, Cd{sup 2+}, and Zn{sup 2+} on alginate gel, native yeast, native yeast immobilized in alginate gel, and caustic-treated yeast immobilized in alginate gel were all compared. Immobilized yeasts could be reactivated and reused in a manner similar to the ion exchange resins. Immobilized caustic-treated yeast has high heavy metal biosorption capacity and high metal removal efficiency in a rather wide acidic pH region. The biosorption isotherm of immobilized caustic-treated yeast wasmore » studied, and empirical equations were obtained. The initial pH of polluted water affected the metal removal efficiency significantly, and the equilibrium biosorption capacity seemed to be temperature independent at lower initial metal concentrations.« less

  19. A pathway of targeted autophagy is induced by DNA damage in budding yeast

    PubMed Central

    Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

    2017-01-01

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

  20. A pathway of targeted autophagy is induced by DNA damage in budding yeast.

    PubMed

    Eapen, Vinay V; Waterman, David P; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G; Loewith, Robbie J; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J; Haber, James E

    2017-02-14

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

  1. Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits*

    PubMed Central

    Hu, Hao; Xu, Yang; Lu, Huang-ping; Xiao, Rui; Zheng, Xiao-dong; Yu, Ting

    2015-01-01

    A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with increasing concentrations of incubated cells. The best concentration was 1×108 cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits. PMID:25845361

  2. Evaluation of yeasts from Tibetan fermented products as agents for biocontrol of blue mold of Nashi pear fruits.

    PubMed

    Hu, Hao; Xu, Yang; Lu, Huang-ping; Xiao, Rui; Zheng, Xiao-dong; Yu, Ting

    2015-04-01

    A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with increasing concentrations of incubated cells. The best concentration was 1×10(8) cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits.

  3. Specialist nectar-yeasts decline with urbanization in Berlin

    NASA Astrophysics Data System (ADS)

    Wehner, Jeannine; Mittelbach, Moritz; Rillig, Matthias C.; Verbruggen, Erik

    2017-03-01

    Nectar yeasts are common inhabitants of insect-pollinated flowers but factors determining their distribution are not well understood. We studied the influence of host identity, environmental factors related to pollution/urbanization, and the distance to a target beehive on local distribution of nectar yeasts within Robinia pseudoacacia L. and Tilia tomentosa Moench in Berlin, Germany. Nectar samples of six individuals per species were collected at seven sites in a 2 km radius from each target beehive and plated on YM-Agar to visualise the different morphotypes, which were then identified by sequencing a section of the 26S rDNA gene. Multivariate linear models were used to analyze the effects of all investigated factors on yeast occurrence per tree. Yeast distribution was mainly driven by host identity. The influence of the environmental factors (NO2, height of construction, soil sealing) strongly depended on the radius around the tree, similar to the distance of the sampled beehive. Incidence of specialist nectar-borne yeast species decreased with increasing pollution/urbanization index. Given that specialist yeast species gave way to generalist yeasts that have a reduced dependency on pollinators for between-flower dispersal, our results indicate that increased urbanization may restrict the movement of nectar-specialized yeasts, via limitations of pollinator foraging behavior.

  4. Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast.

    PubMed

    Bartish, Galyna; Moradi, Hossein; Nygård, Odd

    2007-10-01

    Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

  5. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

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

    USDA-ARS?s Scientific Manuscript database

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

  7. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

    PubMed

    Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

    2003-10-01

    Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

  8. A simple genetic incompatibility causes hybrid male sterility in mimulus.

    PubMed

    Sweigart, Andrea L; Fishman, Lila; Willis, John H

    2006-04-01

    Much evidence has shown that postzygotic reproductive isolation (hybrid inviability or sterility) evolves by the accumulation of interlocus incompatibilities between diverging populations. Although in theory only a single pair of incompatible loci is needed to isolate species, empirical work in Drosophila has revealed that hybrid fertility problems often are highly polygenic and complex. In this article we investigate the genetic basis of hybrid sterility between two closely related species of monkeyflower, Mimulus guttatus and M. nasutus. In striking contrast to Drosophila systems, we demonstrate that nearly complete hybrid male sterility in Mimulus results from a simple genetic incompatibility between a single pair of heterospecific loci. We have genetically mapped this sterility effect: the M. guttatus allele at the hybrid male sterility 1 (hms1) locus acts dominantly in combination with recessive M. nasutus alleles at the hybrid male sterility 2 (hms2) locus to cause nearly complete hybrid male sterility. In a preliminary screen to find additional small-effect male sterility factors, we identified one additional locus that also contributes to some of the variation in hybrid male fertility. Interestingly, hms1 and hms2 also cause a significant reduction in hybrid female fertility, suggesting that sex-specific hybrid defects might share a common genetic basis. This possibility is supported by our discovery that recombination is reduced dramatically in a cross involving a parent with the hms1-hms2 incompatibility.

  9. FTIR microspectroscopy for rapid screening and monitoring of polyunsaturated fatty acid production in commercially valuable marine yeasts and protists.

    PubMed

    Vongsvivut, Jitraporn; Heraud, Philip; Gupta, Adarsha; Puri, Munish; McNaughton, Don; Barrow, Colin J

    2013-10-21

    The increase in polyunsaturated fatty acid (PUFA) consumption has prompted research into alternative resources other than fish oil. In this study, a new approach based on focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopy and multivariate data analysis was developed for the characterisation of some marine microorganisms. Cell and lipid compositions in lipid-rich marine yeasts collected from the Australian coast were characterised in comparison to a commercially available PUFA-producing marine fungoid protist, thraustochytrid. Multivariate classification methods provided good discriminative accuracy evidenced from (i) separation of the yeasts from thraustochytrids and distinct spectral clusters among the yeasts that conformed well to their biological identities, and (ii) correct classification of yeasts from a totally independent set using cross-validation testing. The findings further indicated additional capability of the developed FPA-FTIR methodology, when combined with partial least squares regression (PLSR) analysis, for rapid monitoring of lipid production in one of the yeasts during the growth period, which was achieved at a high accuracy compared to the results obtained from the traditional lipid analysis based on gas chromatography. The developed FTIR-based approach when coupled to programmable withdrawal devices and a cytocentrifugation module would have strong potential as a novel online monitoring technology suited for bioprocessing applications and large-scale production.

  10. Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kere, J.; Grzeschik, K.H.; Limon, J.

    1993-05-01

    Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids the authors mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene. 32 refs., 3 figs., 1 tab.

  11. Prions in Yeast

    PubMed Central

    Liebman, Susan W.; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

  12. Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers.

    PubMed

    Slininger, Patricia J; Dien, Bruce S; Kurtzman, Cletus P; Moser, Bryan R; Bakota, Erica L; Thompson, Stephanie R; O'Bryan, Patricia J; Cotta, Michael A; Balan, Venkatesh; Jin, Mingjie; Sousa, Leonardo da Costa; Dale, Bruce E

    2016-08-01

    Oleaginous yeasts can convert sugars to lipids with fatty acid profiles similar to those of vegetable oils, making them attractive for production of biodiesel. Lignocellulosic biomass is an attractive source of sugars for yeast lipid production because it is abundant, potentially low cost, and renewable. However, lignocellulosic hydrolyzates are laden with byproducts which inhibit microbial growth and metabolism. With the goal of identifying oleaginous yeast strains able to convert plant biomass to lipids, we screened 32 strains from the ARS Culture Collection, Peoria, IL to identify four robust strains able to produce high lipid concentrations from both acid and base-pretreated biomass. The screening was arranged in two tiers using undetoxified enzyme hydrolyzates of ammonia fiber expansion (AFEX)-pretreated cornstover as the primary screening medium and acid-pretreated switch grass as the secondary screening medium applied to strains passing the primary screen. Hydrolyzates were prepared at ∼18-20% solids loading to provide ∼110 g/L sugars at ∼56:39:5 mass ratio glucose:xylose:arabinose. A two stage process boosting the molar C:N ratio from 60 to well above 400 in undetoxified switchgrass hydrolyzate was optimized with respect to nitrogen source, C:N, and carbon loading. Using this process three strains were able to consume acetic acid and nearly all available sugars to accumulate 50-65% of cell biomass as lipid (w/w), to produce 25-30 g/L lipid at 0.12-0.22 g/L/h and 0.13-0.15 g/g or 39-45% of the theoretical yield at pH 6 and 7, a performance unprecedented in lignocellulosic hydrolyzates. Three of the top strains have not previously been reported for the bioconversion of lignocellulose to lipids. The successful identification and development of top-performing lipid-producing yeast in lignocellulose hydrolyzates is expected to advance the economic feasibility of high quality biodiesel and jet fuels from renewable biomass, expanding the market

  13. Band structure calculations of CuAlO2, CuGaO2, CuInO2, and CuCrO2 by screened exchange

    NASA Astrophysics Data System (ADS)

    Gillen, Roland; Robertson, John

    2011-07-01

    We report density functional theory band structure calculations on the transparent conducting oxides CuAlO2, CuGaO2, CuInO2, and CuCrO2. The use of the hybrid functional screened-exchange local density approximation (sX-LDA) leads to considerably improved electronic properties compared to standard LDA and generalized gradient approximation (GGA) approaches. We show that the resulting electronic band gaps compare well with experimental values and previous quasiparticle calculations, and show the correct trends with respect to the atomic number of the cation (Al, Ga, In). The resulting energetic depths of Cu d and O p levels and the valence-band widths are considerable improvements compared to LDA and GGA and are in good agreement with available x-ray photoelectron spectroscopy data. Lastly, we show the calculated imaginary part of the dielectric function for all four systems.

  14. 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.

  15. Stabilization process in Saccharomyces intra and interspecific hybrids in fermentative conditions.

    PubMed

    Pérez-Través, Laura; Lopes, Christian A; Barrio, Eladio; Querol, Amparo

    2014-12-01

    We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30-50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  16. A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment.

    PubMed

    Nardi, Tiziana; Carlot, Milena; De Bortoli, Elena; Corich, Viviana; Giacomini, Alessio

    2006-11-01

    During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.

  17. Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics.

    PubMed

    Ikushima, Shigehito; Tateishi, Yoshiyuki; Kanai, Keiko; Shimada, Emiko; Tanaka, Misa; Ishiguro, Tatsuji; Mizutani, Satoru; Kobayashi, Osamu

    2012-04-01

    Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Screening for estrogenic and antiestrogenic activities of plants growing in Egypt and Thailand

    PubMed Central

    El-Halawany, Ali M.; El Dine, Riham Salah; Chung, Mi Hwa; Nishihara, Tsutomu; Hattori, Masao

    2011-01-01

    Background: There is a growing demand for the discovery of new phytoestrogens to be used as a safe and effective hormonal replacement therapy. Materials and Methods: The methanol extracts of 40 plants from the Egyptian and Thailand folk medicines were screened for their estrogen agonist and antagonist activities. The estrogenic and antiestrogenic effects of the tested extracts were carried out using the yeast two-hybrid assay system expressing ERα and ERβ. In addition, all the extracts were subjected to a naringinase treatment and retested for their estrogenic activity. Results: The methanol extracts of Derris reticulata and Dracaena lourieri showed the most potent estrogenic activity on both estrogen-receptor subtypes, while, the methanol extracts of Butea monosperma, Erythrina fusca, and Dalbergia candenatensis revealed significant estrogenic activity on ERβ only. Nigella sativa, Sophora japonica, Artabotrys harmandii, and Clitorea hanceana showed estrogenic effect only after naringinase treatment. The most potent antiestrogenic effect was revealed by Aframomum melegueta, Dalbergia candenatensis, Dracena loureiri, and Mansonia gagei. PMID:21772754

  19. Differential stress response of Saccharomyces hybrids revealed by monitoring Hsp104 aggregation and disaggregation.

    PubMed

    Kempf, Claudia; Lengeler, Klaus; Wendland, Jürgen

    2017-07-01

    Proteotoxic stress may occur upon exposure of yeast cells to different stress conditions. The induction of stress response mechanisms is important for cells to adapt to changes in the environment and ensure survival. For example, during exposure to elevated temperatures the expression of heat shock proteins such as Hsp104 is induced in yeast. Hsp104 extracts misfolded proteins from aggregates to promote their refolding. We used an Hsp104-GFP reporter to analyze the stress profiles of Saccharomyces species hybrids. To this end a haploid S. cerevisiae strain, harboring a chromosomal HSP104-GFP under control of its endogenous promoter, was mated with stable haploids of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum. Stress response behaviors in these hybrids were followed over time by monitoring the appearance and dissolution of Hsp104-GFP foci upon heat shock. General stress tolerance of these hybrids was related to the growth rate detected during exposure to e.g. ethanol and oxidizing agents. We observed that hybrids were generally more resistant to high temperature and ethanol stress compared to their parental strains. Amongst the hybrids differential responses regarding the appearance of Hsp104-foci and the time required for dissolving these aggregates were observed. The S. cerevisiae/S. paradoxus hybrid, combining the two most closely related strains, performed best under these conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076

  1. Classification of yeast cells from image features to evaluate pathogen conditions

    NASA Astrophysics Data System (ADS)

    van der Putten, Peter; Bertens, Laura; Liu, Jinshuo; Hagen, Ferry; Boekhout, Teun; Verbeek, Fons J.

    2007-01-01

    Morphometrics from images, image analysis, may reveal differences between classes of objects present in the images. We have performed an image-features-based classification for the pathogenic yeast Cryptococcus neoformans. Building and analyzing image collections from the yeast under different environmental or genetic conditions may help to diagnose a new "unseen" situation. Diagnosis here means that retrieval of the relevant information from the image collection is at hand each time a new "sample" is presented. The basidiomycetous yeast Cryptococcus neoformans can cause infections such as meningitis or pneumonia. The presence of an extra-cellular capsule is known to be related to virulence. This paper reports on the approach towards developing classifiers for detecting potentially more or less virulent cells in a sample, i.e. an image, by using a range of features derived from the shape or density distribution. The classifier can henceforth be used for automating screening and annotating existing image collections. In addition we will present our methods for creating samples, collecting images, image preprocessing, identifying "yeast cells" and creating feature extraction from the images. We compare various expertise based and fully automated methods of feature selection and benchmark a range of classification algorithms and illustrate successful application to this particular domain.

  2. Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii

    PubMed Central

    Douradinha, Bruno; Reis, Viviane CB; Rogers, Matthew B; Torres, Fernando AG; Evans, Jared D; Marques Jr, Ernesto TA

    2014-01-01

    Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (>96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest. PMID:24013355

  3. Novel insights in genetic transformation of the probiotic yeast Saccharomyces boulardii.

    PubMed

    Douradinha, Bruno; Reis, Viviane C B; Rogers, Matthew B; Torres, Fernando A G; Evans, Jared D; Marques, Ernesto T A

    2014-01-01

    Saccharomyces boulardii (S. boulardii) is a probiotic yeast related to Saccharomyces cerevisiae (S. cerevisiae) but with distinct genetic, taxonomic and metabolic properties. S. cerevisiae has been used extensively in biotechnological applications. Currently, many strains are available, and multiple genetic tools have been developed, which allow the expression of several exogenous proteins of interest with applications in the fields of medicine, biofuels, the food industry, and scientific research, among others. Although S. boulardii has been widely studied due to its probiotic properties against several gastrointestinal tract disorders, very few studies addressed the use of this yeast as a vector for expression of foreign genes of interest with biotechnological applications. Here we show that, despite the similarity of the two yeasts, not all genetic tools used in S. cerevisiae can be applied in S. boulardii. While transformation of the latter could be obtained using a commercial kit developed for the former, consequent screening of successful transformants had to be optimized. We also show that several genes frequently used in genetic manipulation of S. cerevisiae (e.g., promoters and resistance markers) are present in S. boulardii. Sequencing revealed a high rate of homology (> 96%) between the orthologs of the two yeasts. However, we also observed some of them are not eligible to be targeted for transformation of S. boulardii. This work has important applications toward the potential of this probiotic yeast as an expression system for genes of interest.

  4. Identification of a new Mpl-interacting protein, Atp5d.

    PubMed

    Liu, Hongyan; Zhao, Zhenhu; Zhong, Yuxu; Shan, Yajun; Sun, Xiaohong; Mao, Bingzhi; Cong, Yuwen

    2014-06-01

    Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

  5. Hierarchical porous carbon/MnO2 hybrids as supercapacitor electrodes.

    PubMed

    Lee, Min Eui; Yun, Young Soo; Jin, Hyoung-Joon

    2014-12-01

    Hybrid electrodes of hierarchical porous carbon (HPC) and manganese oxide (MnO2) were synthesized using a fast surface redox reaction of potassium permanganate under facile immersion methods. The HPC/MnO2 hybrids had a number of micropores and macropores and the MnO2 nanoparticles acted as a pseudocapacitive material. The synergistic effects of electric double-layer capacitor (EDLC)-induced capacitance and pseudocapacitance brought about a better electrochemical performance of the HPC/MnO2 hybrid electrodes compared to that obtained with a single component. The hybrids showed a specific capacitance of 228 F g(-1) and good cycle stability over 1000 cycles.

  6. High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

    PubMed

    Back, Alexandre; Rossignol, Tristan; Krier, François; Nicaud, Jean-Marc; Dhulster, Pascal

    2016-08-23

    Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. To our knowledge, this is the

  7. Crack-healing function of metal/Al2O3 hybrid materials

    NASA Astrophysics Data System (ADS)

    Nanko, M.; Maruoka, D.; Nguyen, T. D.

    2011-10-01

    Nano-Ni/Al2O3 hybrid materials have the crack-healing function by thermal oxidation process such as 1200°C for 6 h in air. In this hybrid material system, crack was filled up by an oxidation product, NiAl2O4, via outward diffusion of cations along grain boundaries of Al2O3 matrix. Ni/Al2O3 with Y2O3 doping and SiC+Ni/Al2O3 nano-hybrid materials have similar crack-healing performance with better oxidation resistance at high temperatures than Ni/Al2O3 nano-hybrid materials. Mo/Al2O3 hybrid materials were studied on a candidate with crack-healing function via thermal oxidation process at temperatures as low as 700°C.

  8. eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

    PubMed Central

    Menacho-Márquez, Mauricio; Rodríguez-Hernández, Carlos J; Villaronga, M Ángeles; Pérez-Valle, Jorge; Gadea, José; Belandia, Borja; Murguía, José R

    2015-01-01

    β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses. PMID:25590579

  9. A combination of 2D similarity search, pharmacophore, and molecular docking techniques for the identification of vascular endothelial growth factor receptor-2 inhibitors.

    PubMed

    Ai, Guanhua; Tian, Caiping; Deng, Dawei; Fida, Guissi; Chen, Haiyan; Ma, Yuxiang; Ding, Li; Gu, Yueqing

    2015-04-01

    The human vascular endothelial growth factor receptor-2 (VEGFR-2) has been an attractive target for the inhibition of angiogenesis. In the current study, we used a hybrid protocol of virtual screening methods to retrieve new VEGFR-2 inhibitors from the Zinc-Specs Database (441 574 compounds). The hybrid protocol included the initial screening of candidates by comparing the 2D similarity to five reported top active inhibitors of 13 VEGFR-2 X-ray crystallography structures, followed by the pharmacophore modeling of virtual screening on the basis of receptor-ligand interactions and further narrowing by LibDOCK to obtain the final hits. Two compounds (AN-919/41439526 and AK-968/40939851) with a high libscore were selected as the final hits for a subsequent cell cytotoxicity study. The two compounds screened exerted significant inhibitory effects on the proliferation of cancer cells (U87 and MCF-7). The results indicated that the hybrid procedure is an effective approach for screening specific receptor inhibitors.

  10. Hastatic order in URu2Si2 : Hybridization with a twist

    NASA Astrophysics Data System (ADS)

    Chandra, Premala; Coleman, Piers; Flint, Rebecca

    2015-05-01

    The broken symmetry that develops below 17.5 K in the heavy fermion compound URu2Si2 has long eluded identification. Here we argue that the recent observation of Ising quasiparticles in URu2Si2 results from a spinor hybridization order parameter that breaks double time-reversal symmetry by mixing states of integer and half-integer spin. Such "hastatic order" (hasta: [Latin] spear) hybridizes Kramers conduction electrons with Ising, non-Kramers 5 f2 states of the uranium atoms to produce Ising quasiparticles. The development of a spinorial hybridization at 17.5 K accounts for both the large entropy of condensation and the magnetic anomaly observed in torque magnetometry. This paper develops the theory of hastatic order in detail, providing the mathematical development of its key concepts. Hastatic order predicts a tiny transverse moment in the conduction sea, a colossal Ising anisotropy in the nonlinear susceptibility anomaly and a resonant energy-dependent nematicity in the tunneling density of states.

  11. Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast

    PubMed Central

    Walther, Andrea; Hesselbart, Ana; Wendland, Jürgen

    2014-01-01

    Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis, which was originally termed Unterhefe No. 1 by Emil Chr. Hansen and has been used in production in since 1883. S. carlsbergensis belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than group II/Frohberg-type lager yeasts, e.g., the Weihenstephan strain WS34/70. Here, we sequenced S. carlsbergensis using next generation sequencing technologies. Lager yeasts are descendants from hybrids formed between a S. cerevisiae parent and a parent similar to S. eubayanus. Accordingly, the S. carlsbergensis 19.5-Mb genome is substantially larger than the 12-Mb S. cerevisiae genome. Based on the sequence scaffolds, synteny to the S. cerevisae genome, and by using directed polymerase chain reaction for gap closure, we generated a chromosomal map of S. carlsbergensis consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within S. carlsbergensis via chromosome loss and loss of heterozygosity specifically of parts derived from the S. cerevisiae parent. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. carlsbergensis. This inferred that this strain is basically triploid with a diploid S. eubayanus and haploid S. cerevisiae genome content. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. cerevisiae and S. eubayanus genomes. Based on conserved translocations between the parental genomes in S. carlsbergensis and the Weihenstephan strain we propose a joint evolutionary ancestry for lager yeast strains. PMID:24578374

  12. HEXT, a software supporting tree-based screens for hybrid taxa in multilocus data sets, and an evaluation of the homoplasy excess test.

    PubMed

    Schneider, Kevin; Koblmüller, Stephan; Sefc, Kristina M

    2015-11-11

    The homoplasy excess test (HET) is a tree-based screen for hybrid taxa in multilocus nuclear phylogenies. Homoplasy between a hybrid taxon and the clades containing the parental taxa reduces bootstrap support in the tree. The HET is based on the expectation that excluding the hybrid taxon from the data set increases the bootstrap support for the parental clades, whereas excluding non-hybrid taxa has little effect on statistical node support. To carry out a HET, bootstrap trees are calculated with taxon-jackknife data sets, that is excluding one taxon (species, population) at a time. Excess increase in bootstrap support for certain nodes upon exclusion of a particular taxon indicates the hybrid (the excluded taxon) and its parents (the clades with increased support).We introduce a new software program, hext, which generates the taxon-jackknife data sets, runs the bootstrap tree calculations, and identifies excess bootstrap increases as outlier values in boxplot graphs. hext is written in r language and accepts binary data (0/1; e.g. AFLP) as well as co-dominant SNP and genotype data.We demonstrate the usefulness of hext in large SNP data sets containing putative hybrids and their parents. For instance, using published data of the genus Vitis (~6,000 SNP loci), hext output supports V. × champinii as a hybrid between V. rupestris and V. mustangensis .With simulated SNP and AFLP data sets, excess increases in bootstrap support were not always connected with the hybrid taxon (false positives), whereas the expected bootstrap signal failed to appear on several occasions (false negatives). Potential causes for both types of spurious results are discussed.With both empirical and simulated data sets, the taxon-jackknife output generated by hext provided additional signatures of hybrid taxa, including changes in tree topology across trees, consistent effects of exclusions of the hybrid and the parent taxa, and moderate (rather than excessive) increases in bootstrap support

  13. A Protein Linkage Map of the P2 Nonstructural Proteins of Poliovirus

    PubMed Central

    Cuconati, Andrea; Xiang, Wenkai; Lahser, Frederick; Pfister, Thomas; Wimmer, Eckard

    1998-01-01

    The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2Apro, 2BC, 2C, and 2B. Specifically, 2Apro can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. trans-dominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341–4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome. PMID:9445030

  14. Cell-in-Shell Hybrids: Chemical Nanoencapsulation of Individual Cells.

    PubMed

    Park, Ji Hun; Hong, Daewha; Lee, Juno; Choi, Insung S

    2016-05-17

    Nature has developed a fascinating strategy of cryptobiosis ("secret life") for counteracting the stressful, and often lethal, environmental conditions that fluctuate sporadically over time. For example, certain bacteria sporulate to transform from a metabolically active, vegetative state to an ametabolic endospore state. The bacterial endospores, encased within tough biomolecular shells, withstand the extremes of harmful stressors, such as radiation, desiccation, and malnutrition, for extended periods of time and return to a vegetative state by breaking their protective shells apart when their environment becomes hospitable for living. Certain ciliates and even higher organisms, for example, tardigrades, and others are also found to adopt a cryptobiotic strategy for survival. A common feature of cryptobiosis is the structural presence of tough sheaths on cellular structures. However, most cells and cellular assemblies are not "spore-forming" and are vulnerable to the outside threats. In particular, mammalian cells, enclosed with labile lipid bilayers, are highly susceptible to in vitro conditions in the laboratory and daily life settings, making manipulation and preservation difficult outside of specialized conditions. The instability of living cells has been a main bottleneck to the advanced development of cell-based applications, such as cell therapy and cell-based sensors. A judicious question arises: can cellular tolerance against harmful stresses be enhanced by simply forming cell-in-shell hybrid structures? Experimental results suggest that the answer is yes. A micrometer-sized "Iron Man" can be generated by chemically forming an ultrathin (<100 nm) but durable shell on a "non-spore-forming" cell. Since the report on silica nanoencapsulation of yeast cells, in which cytoprotective yeast-in-silica hybrids were formed, several synthetic strategies have been developed to encapsulate individual cells in a cytocompatible fashion, mimicking the cryptobiotic cell

  15. Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii.

    PubMed

    Naumov, Gennadi I; Lee, Ching-Fu; Naumova, Elena S

    2013-01-01

    Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.

  16. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

    PubMed Central

    Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

    2012-01-01

    A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

  17. Isolation of proflavine as a blocker of G protein-gated inward rectifier potassium channels by a cell growth-based screening system.

    PubMed

    Kawada, Hitoshi; Inanobe, Atsushi; Kurachi, Yoshihisa

    2016-10-01

    The overexpression of Kir3.2, a subunit of the G protein-gated inwardly rectifying K(+) channel, is implicated in some of the neurological phenotypes of Down syndrome (DS). Chemical compounds that block Kir3.2 are expected to improve the symptoms of DS. The purpose of this study is to develop a cell-based screening system to identify Kir3.2 blockers and then investigate the mode of action of the blocker. Chemical screening was carried out using a K(+) transporter-deficient yeast strain that expressed a constitutively active Kir3.2 mutant. The mode of action of an effective blocker was electrophysiologically analyzed using Kir channels expressed in Xenopus oocytes. Proflavine was identified to inhibit the growth of Kir3.2-transformant cells and Kir3.2 activity in a concentration-dependent manner. The current inhibition was strong when membrane potentials (Vm) was above equilibrium potential of K(+) (EK). When Vm was below EK, the blockage apparently depended on the difference between Vm and [K(+)]. Furthermore, the inhibition became stronger by lowering extracellular [K(+)]. These results indicated that the yeast strain serves as a screening system to isolate Kir3.2 blockers and proflavine is a prototype of a pore blocker of Kir3.2. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Construction of human chromosome 21-specific yeast artificial chromosomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McCormick, M.K.; Shero, J.H.; Hieter, P.A.

    1989-12-01

    Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to > 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtainedmore » from a mouse-human hybrid, ranging in size from 200 to > 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from {approx} 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.« less

  19. L-arabinose fermenting yeast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko

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

  20. L-arabinose fermenting yeast

    DOEpatents

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

    2014-09-23

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

  1. Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

    PubMed Central

    Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

    1993-01-01

    Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

  2. High Surface Area MoS 2/Graphene Hybrid Aerogel for Ultrasensitive NO 2 Detection

    DOE PAGES

    Long, Hu; Harley-Trochimczyk, Anna; Pham, Thang; ...

    2016-05-23

    A MoS 2/graphene hybrid aerogel synthesized with two-dimensional MoS 2 sheets coating a high surface area graphene aerogel scaffold is characterized and used for ultrasensitive NO 2 detection. The combination of graphene and MoS 2 leads to improved sensing properties with the graphene scaffold providing high specific surface area and high electrical and thermal conductivity and the single to few-layer MoS2 sheets providing high sensitivity and selectivity to NO 2. The hybrid aerogel is integrated onto a low-power microheater platform to probe the gas sensing performance. At room temperature, the sensor exhibits an ultralow detection limit of 50 ppb NOmore » 2. By heating the material to 200 °C, the response and recovery times to reach 90% of the final signal decrease to <1 min, while retaining the low detection limit. The MoS 2/graphene hybrid also shows good selectivity for NO 2 against H 2 and CO, especially when compared to bare graphene aerogel. The unique structure of the hybrid aerogel is responsible for the ultrasensitive, selective, and fast NO 2 sensing. The improved sensing performance of this hybrid aerogel also suggests the possibility of other 2D material combinations for further sensing applications.« less

  3. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

    PubMed Central

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-01-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  4. Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species.

    PubMed

    Sitepu, Irnayuli R; Sestric, Ryan; Ignatia, Laura; Levin, David; German, J Bruce; Gillies, Laura A; Almada, Luis A G; Boundy-Mills, Kyria L

    2013-09-01

    Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeasts species

    PubMed Central

    Sitepu, Irnayuli R.; Sestric, Ryan; Ignatia, Laura; Levin, David; German, J. Bruce; Gillies, Laura A.; Almada, Luis A.G.; Boundy-Mills, Kyria L.

    2013-01-01

    Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified. PMID:23891835

  6. Enhancement of the Initial Rate of Ethanol Fermentation Due to Dysfunction of Yeast Stress Response Components Msn2p and/or Msn4p▿ †

    PubMed Central

    Watanabe, Daisuke; Wu, Hong; Noguchi, Chiemi; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

    2011-01-01

    Sake yeasts (strains of Saccharomyces cerevisiae) produce high concentrations of ethanol in sake fermentation. To investigate the molecular mechanisms underlying this brewing property, we compared gene expression of sake and laboratory yeasts in sake mash. DNA microarray and reporter gene analyses revealed defects of sake yeasts in environmental stress responses mediated by transcription factors Msn2p and/or Msn4p (Msn2/4p) and stress response elements (STRE). Furthermore, we found that dysfunction of MSN2 and/or MSN4 contributes to the higher initial rate of ethanol fermentation in both sake and laboratory yeasts. These results provide novel insights into yeast stress responses as major impediments of effective ethanol fermentation. PMID:21131516

  7. Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

    PubMed Central

    Lowes, K. F.; Shearman, C. A.; Payne, J.; MacKenzie, D.; Archer, D. B.; Merry, R. J.; Gasson, M. J.

    2000-01-01

    The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts. PMID:10698773

  8. Production of Sophorolipid from an Identified Current Yeast, Lachancea thermotolerans BBMCZ7FA20, Isolated from Honey Bee.

    PubMed

    Mousavi, Fereshteh; Beheshti-Maal, Keivan; Massah, Ahmadreza

    2015-08-01

    Biosurfactants are a family of diverse amphipathic molecules that are produced by several microorganisms such as bacteria, molds, and yeasts. These surface active agents have several applications in agriculture, oil processing, food, and pharmaceutical industries. In this research using YMG and YUG culture media, a native yeast strain, HG5, was isolated from honey bee. The oil spread test as a screening method was used to evaluate biosurfactant production by the yeast HG5 isolate. The 5.8s-rDNA analysis confirmed that the isolated yeast was related to Lachancea thermotolerans. We named this strain Lachancea thermotolerans strain BBMCZ7FA20 and its 5.8s-rDNA sequence was deposited in GenBank, NCBI under accession number of KM042082.1. The best precursor of biosurfactant production was canola oil and the sophorolipid amount was measured for 24.2 g/l. The thin layer chromatography and Fourier Transform Infrared Spectroscopy analysis showed that the extracted biosurfactant from Lachancea thermotolerans was sophorolipid. In conclusion, this is the first report of sophorolipid production by a native yeast Lachancea thermotolerans BBMCZ7FA20 we isolated from the honey bee gut collected from an apiary farm in Saman, Chaharmahal Bakhtiari province, Iran. We suggested that some cost-effective supplements such as canola oil, sunflower oil, and corn oils could be applied for increasing the sophorolipid production by this native yeast strain. According to several applications of biosurfactants in today world, the production of sophorolipid by Lachancea thermotolerans could be considered as a potential in the current industrial microbiology and modern microbial biotechnology.

  9. Effects of dispersible MoS2 nanosheets and Nano-silver coexistence on the metabolome of yeast.

    PubMed

    Yang, Qi; Zhang, Lei; Ben, Ailing; Wu, Na; Yi, Yanliang; Jiang, Ling; Huang, He; Yu, Yadong

    2018-05-01

    As a new rising star in the post-graphene two-dimensional materials (2DMs), molybdenum disulfide (MoS 2 ) attracts increasing attentions and is widely applied. However, the chemical and toxicological interaction between MoS 2 and other co-contaminants is still poorly understood. Nano-silver (N-Ag) is the most commonly used nanomaterial in commercial products and distributed widely in the environment. Herein, we investigated the effects of chitosan functionalized MoS 2 (CS-MoS 2 ) nanosheets, a water-dispersible form of MoS 2 , on the microbial toxicity of N-Ag. We found that the incorporation of CS-MoS 2 nanosheets attenuated the oxidative stress induced by N-Ag on yeast cells, while caused more membrane stress. In addition, the inhibition of N-Ag on the metabolic activities of yeast cells could be attenuated by CS-MoS 2 nanosheets as well. The coexistence of N-Ag and CS-MoS 2 nanosheets mainly perturbed the amino acid-related metabolic pathways in yeast cells, and phosphoric acid was a potential nanotoxicity biomarker. We further found that CS-MoS 2 nanosheets dramatically absorbed the Ag ion released from N-Ag, which might be responsible for its attenuation effect on the microbial toxicity of N-Ag. Our findings provide more new insights for the ecotoxicity evaluation of MoS 2 and other 2DMs. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Tolerant industrial yeast Saccharomyces cerevisiae posses a more robust cell wall integrity signaling pathway against 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde.

    PubMed

    Liu, Z Lewis; Wang, Xu; Weber, Scott A

    2018-06-20

    Cell wall integrity signaling pathway in Saccharomyces cerevisiae is a conserved function for detecting and responding to cell stress conditions but less understood for industrial yeast. We examined gene expression dynamics for a tolerant industrial yeast strain NRRL Y-50049 in response to challenges of furfural and HMF through comparative quantitative gene expression analysis using pathway-based qRT-PCR array assays. All tested genes from Y-50049, except for MLP2, demonstrated more resistant and significantly increased gene expression than that from a laboratory strain BY4741. While all five sensor encoding genes WSC1, WSC2, WSC3, MID2 and MTL1 from both strains were activated in response to the furfural-HMF treatment, WSC3 from Y-50049 demonstrated the most increased expression over time compared with any other sensor genes. These results suggested the industrial yeast poses more robust cell wall integrity pathway, and gene WSC3 could have the special capability for signal transmission against furfural and HMF. Among five single nucleotide variations discovered in WSC3 from Y-50049, three were found to be non-synonymous mutations resulting in amino acid alterations of Ser 158  → Tyr 158 , Val 186  → Ile 186 , and Glu 430  → Asp 430 . Our results suggest the industrial yeast as a more desirable delivery vehicle for the next-generation biocatalyst development. Published by Elsevier B.V.

  11. Carbon nanotube TiO2 hybrid films for detecting traces of O2

    NASA Astrophysics Data System (ADS)

    Llobet, E.; Espinosa, E. H.; Sotter, E.; Ionescu, R.; Vilanova, X.; Torres, J.; Felten, A.; Pireaux, J. J.; Ke, X.; Van Tendeloo, G.; Renaux, F.; Paint, Y.; Hecq, M.; Bittencourt, C.

    2008-09-01

    Hybrid titania films have been prepared using an adapted sol-gel method for obtaining well-dispersed hydrogen plasma-treated multiwall carbon nanotubes in either pure titania or Nb-doped titania. The drop-coating method has been used to fabricate resistive oxygen sensors based on titania or on titania and carbon nanotube hybrids. Morphology and composition studies have revealed that the dispersion of low amounts of carbon nanotubes within the titania matrix does not significantly alter its crystallization behaviour. The gas sensitivity studies performed on the different samples have shown that the hybrid layers based on titania and carbon nanotubes possess an unprecedented responsiveness towards oxygen (i.e. more than four times higher than that shown by optimized Nb-doped TiO2 films). Furthermore, hybrid sensors containing carbon nanotubes respond at significantly lower operating temperatures than their non-hybrid counterparts. These new hybrid sensors show a strong potential for monitoring traces of oxygen (i.e. <=10 ppm) in a flow of CO2, which is of interest for the beverage industry.

  12. A Simple Genetic Incompatibility Causes Hybrid Male Sterility in Mimulus

    PubMed Central

    Sweigart, Andrea L.; Fishman, Lila; Willis, John H.

    2006-01-01

    Much evidence has shown that postzygotic reproductive isolation (hybrid inviability or sterility) evolves by the accumulation of interlocus incompatibilities between diverging populations. Although in theory only a single pair of incompatible loci is needed to isolate species, empirical work in Drosophila has revealed that hybrid fertility problems often are highly polygenic and complex. In this article we investigate the genetic basis of hybrid sterility between two closely related species of monkeyflower, Mimulus guttatus and M. nasutus. In striking contrast to Drosophila systems, we demonstrate that nearly complete hybrid male sterility in Mimulus results from a simple genetic incompatibility between a single pair of heterospecific loci. We have genetically mapped this sterility effect: the M. guttatus allele at the hybrid male sterility 1 (hms1) locus acts dominantly in combination with recessive M. nasutus alleles at the hybrid male sterility 2 (hms2) locus to cause nearly complete hybrid male sterility. In a preliminary screen to find additional small-effect male sterility factors, we identified one additional locus that also contributes to some of the variation in hybrid male fertility. Interestingly, hms1 and hms2 also cause a significant reduction in hybrid female fertility, suggesting that sex-specific hybrid defects might share a common genetic basis. This possibility is supported by our discovery that recombination is reduced dramatically in a cross involving a parent with the hms1–hms2 incompatibility. PMID:16415357

  13. Characterization of winemaking yeast by cell number-size distribution analysis through flow field-flow fractionation with multi-wavelength turbidimetric detection.

    PubMed

    Zattoni, Andrea; Melucci, Dora; Reschiglian, Pierluigi; Sanz, Ramsés; Puignou, Lluís; Galceran, Maria Teresa

    2004-10-29

    Yeasts are widely used in several areas of food industry, e.g. baking, beer brewing, and wine production. Interest in new analytical methods for quality control and characterization of yeast cells is thus increasing. The biophysical properties of yeast cells, among which cell size, are related to yeast cell capabilities to produce primary and secondary metabolites during the fermentation process. Biophysical properties of winemaking yeast strains can be screened by field-flow fractionation (FFF). In this work we present the use of flow FFF (FlFFF) with turbidimetric multi-wavelength detection for the number-size distribution analysis of different commercial winemaking yeast varieties. The use of a diode-array detector allows to apply to dispersed samples like yeast cells the recently developed method for number-size (or mass-size) analysis in flow-assisted separation techniques. Results for six commercial winemaking yeast strains are compared with data obtained by a standard method for cell sizing (Coulter counter). The method here proposed gives, at short analysis time, accurate information on the number of cells of a given size, and information on the total number of cells.

  14. Uranium bioprecipitation mediated by yeasts utilizing organic phosphorus substrates.

    PubMed

    Liang, Xinjin; Csetenyi, Laszlo; Gadd, Geoffrey Michael

    2016-06-01

    In this research, we have demonstrated the ability of several yeast species to mediate U(VI) biomineralization through uranium phosphate biomineral formation when utilizing an organic source of phosphorus (glycerol 2-phosphate disodium salt hydrate (C3H7Na2O6P·xH2O (G2P)) or phytic acid sodium salt hydrate (C6H18O24P6·xNa(+)·yH2O (PyA))) in the presence of soluble UO2(NO3)2. The formation of meta-ankoleite (K2(UO2)2(PO4)2·6(H2O)), chernikovite ((H3O)2(UO2)2(PO4)2·6(H2O)), bassetite (Fe(++)(UO2)2(PO4)2·8(H2O)), and uramphite ((NH4)(UO2)(PO4)·3(H2O)) on cell surfaces was confirmed by X-ray diffraction in yeasts grown in a defined liquid medium amended with uranium and an organic phosphorus source, as well as in yeasts pre-grown in organic phosphorus-containing media and then subsequently exposed to UO2(NO3)2. The resulting minerals depended on the yeast species as well as physico-chemical conditions. The results obtained in this study demonstrate that phosphatase-mediated uranium biomineralization can occur in yeasts supplied with an organic phosphate substrate as sole source of phosphorus. Further understanding of yeast interactions with uranium may be relevant to development of potential treatment methods for uranium waste and utilization of organic phosphate sources and for prediction of microbial impacts on the fate of uranium in the environment.

  15. Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows.

    PubMed

    Chung, Y-H; Walker, N D; McGinn, S M; Beauchemin, K A

    2011-05-01

    Fifteen ruminally cannulated, nonlactating Holstein cows were used to measure the effects of 2 strains of Saccharomyces cerevisiae, fed as active dried yeasts, on ruminal pH and fermentation and enteric methane (CH(4)) emissions. Nonlactating cows were blocked by total duration (h) that their ruminal pH was below 5.8 during a 6-d pre-experimental period. Within each block, cows were randomly assigned to control (no yeast), yeast strain 1 (Levucell SC), or yeast strain 2 (a novel strain selected for enhanced in vitro fiber degradation), with both strains (Lallemand Animal Nutrition, Montréal, QC, Canada) providing 1 × 10(10) cfu/head per day. Cows were fed once daily a total mixed ration consisting of a 50:50 forage to concentrate ratio (dry matter basis). The yeast strains were dosed via the rumen cannula daily at the time of feeding. During the 35-d experiment, ruminal pH was measured continuously for 7 d (d 22 to 28) by using an indwelling system, and CH(4) gas was measured for 4 d (d 32 to 35) using the sulfur hexafluoride tracer gas technique (with halters and yokes). Rumen contents were sampled on 2 d (d 22 and 26) at 0, 3, and 6h after feeding. Dry matter intake, body weight, and apparent total-tract digestibility of nutrients were not affected by yeast feeding. Strain 2 decreased the average daily minimum (5.35 vs. 5.65 or 5.66), mean (5.98 vs. 6.24 or 6.34), and maximum ruminal pH (6.71 vs. 6.86 or 6.86), and prolonged the time that ruminal pH was below 5.8 (7.5 vs. 3.3 or 1.0 h/d) compared with the control or strain 1, respectively. The molar percentage of acetate was lower and that of propionate was greater in the ruminal fluid of cows receiving strain 2 compared with cows receiving no yeast or strain 1. Enteric CH(4) production adjusted for intake of dry matter or gross energy, however, did not differ between either yeast strain compared with the control but it tended to be reduced by 10% when strain 2 was compared with strain 1. The study shows that

  16. Competitive Genomic Screens of Barcoded Yeast Libraries

    PubMed Central

    Urbanus, Malene; Proctor, Michael; Heisler, Lawrence E.; Giaever, Guri; Nislow, Corey

    2011-01-01

    By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment. PMID:21860376

  17. Indigenous Georgian Wine-Associated Yeasts and Grape Cultivars to Edit the Wine Quality in a Precision Oenology Perspective.

    PubMed

    Vigentini, Ileana; Maghradze, David; Petrozziello, Maurizio; Bonello, Federica; Mezzapelle, Vito; Valdetara, Federica; Failla, Osvaldo; Foschino, Roberto

    2016-01-01

    In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' = 0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (°Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated

  18. Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory▿

    PubMed Central

    Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

    2007-01-01

    Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

  19. Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate.

    PubMed

    Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

    2016-03-01

    In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.

  20. Genomic reconstruction to improve bioethanol and ergosterol production of industrial yeast Saccharomyces cerevisiae.

    PubMed

    Zhang, Ke; Tong, Mengmeng; Gao, Kehui; Di, Yanan; Wang, Pinmei; Zhang, Chunfang; Wu, Xuechang; Zheng, Daoqiong

    2015-02-01

    Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.

  1. In vitro profiling of toxicity and endocrine disrupting effects of bisphenol analogues by employing MCF-7 cells and two-hybrid yeast bioassay.

    PubMed

    Lei, Bingli; Xu, Jie; Peng, Wei; Wen, Yu; Zeng, Xiangying; Yu, Zhiqiang; Wang, Yipei; Chen, Tian

    2017-01-01

    The potentially adverse health implications of bisphenol A (BPA) have led to increasing use of alternative bisphenols (BPs). However, little is known about the toxicity of alternative BPs. In this study, the cytotoxicity, genotoxicity, intracellular ROS formation, and Ca 2+ fluctuation effects of BPs on MCF-7 cells were evaluated. At the same time, the estrogenic and thyroidal hormone effect potentials of six BPs were also evaluated using two-hybrid yeast bioassay. The results showed that most BPs at 0.01-1 μM significantly increased cell viability in MCF-7 cells and at higher exposure concentrations of 25-100 μM, they caused a significant decrease of cell viability. At the same time, these BPs also at 25-100 μM significantly increased LDH release of MCF-7 cells. In addition, several BPs at 10-50 μM resulted in a significantly concentration-depended increase in DNA-damaging effect on MCF-7 cells and elevated ROS production. Most BPs at 0.0001-10 μM significantly increased intracellular Ca 2+ level. These results showed that bisphenol AF (BPAF) and thiodiphenol (TDP) exerted cell biological effect, estrogenic, and thyroidal effect potentials greater than those of BPA. The cytotoxicity and endocrine disrupting effects of other BPs are similar to or slightly lower than those of BPA. Therefore, as potential alternatives to BPA, endocrine disrupting effects and potential health harm of alternative BPs to human can also not be ignored. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 278-289, 2017. © 2016 Wiley Periodicals, Inc.

  2. Yeast two-hybrid and pull-down assays propose an interaction between P50 of apple chlorotic leaf spot virus and PR-10 of Malus sylvestris cv. R12740-7A.

    PubMed

    Wang, Y; Li, N; Zhao, X; Hu, J; He, Y; Hu, T; Wang, S; Wang, Y; Cao, K

    Apple chlorotic leaf spot virus (ACLSV) movement protein (P50) is involved in cell-to-cell transport and influences the long-distance spread of silencing activity. Previously, we obtained 69 P50-interacting proteins from Malus sylvestris cv. R12740-7A and using bioinformatics analyzed their biological functions. In this study, we used the GAL4-based two-hybrid yeast system and His pull-down assays to confirm an interaction between PR-10 of M. sylvestris cv. R12740-7A and ACLSV P50. Our results provide a theoretical basis for further research on the biological function of PR-10 in ACLSV infection and the interacting mechanism between host and virus.

  3. A stable hybrid containing haploid genomes of two obligate diploid Candida species.

    PubMed

    Chakraborty, Uttara; Mohamed, Aiyaz; Kakade, Pallavi; Mugasimangalam, Raja C; Sadhale, Parag P; Sanyal, Kaustuv

    2013-08-01

    Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

  4. Selection of Yeast Strains for Tequila Fermentation Based on Growth Dynamics in Combined Fructose and Ethanol Media.

    PubMed

    Aldrete-Tapia, J A; Miranda-Castilleja, D E; Arvizu-Medrano, S M; Hernández-Iturriaga, M

    2018-02-01

    The high concentration of fructose in agave juice has been associated with reduced ethanol tolerance of commercial yeasts used for tequila production and low fermentation yields. The selection of autochthonous strains, which are better adapted to agave juice, could improve the process. In this study, a 2-step selection process of yeasts isolated from spontaneous fermentations for tequila production was carried out based on analysis of the growth dynamics in combined conditions of high fructose and ethanol. First, yeast isolates (605) were screened to identify strains tolerant to high fructose (20%) and to ethanol (10%), yielding 89 isolates able to grow in both conditions. From the 89 isolates, the growth curves under 8 treatments of combined fructose (from 20% to 5%) and ethanol (from 0% to 10%) were obtained, and the kinetic parameters were analyzed with principal component analysis and k-means clustering. The resulting yeast strain groups corresponded to the fast, medium and slow growers. A second clustering of only the fast growers led to the selection of 3 Saccharomyces strains (199, 230, 231) that were able to grow rapidly in 4 out of the 8 conditions evaluated. This methodology differentiated strains phenotypically and could be further used for strain selection in other processes. A method to select yeast strains for fermentation taking into account the natural differences of yeast isolates. This methodology is based on the cell exposition to combinations of sugar and ethanol, which are the most important stress factors in fermentation. This strategy will help to identify the most tolerant strain that could improve ethanol yield and reduce fermentation time. © 2018 Institute of Food Technologists®.

  5. The Spheroplast Lysis Assay for Yeast in Microtiter Plate Format

    PubMed Central

    Ovalle, Rafael; Spencer, Moyah; Thiwanont, Monthiwa; Lipke, Peter N.

    1999-01-01

    A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes. PMID:10427014

  6. Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations

    PubMed Central

    Meersman, Esther; Steensels, Jan; Paulus, Tinneke; Struyf, Nore; Saels, Veerle; Mathawan, Melissa; Koffi, Jean; Vrancken, Gino

    2015-01-01

    Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production. PMID:26150457

  7. Differential Adsorption of Ochratoxin A and Anthocyanins by Inactivated Yeasts and Yeast Cell Walls during Simulation of Wine Aging

    PubMed Central

    Petruzzi, Leonardo; Baiano, Antonietta; De Gianni, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria; Bevilacqua, Antonio

    2015-01-01

    The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify the intensity of the phenomenon. The aim of this study was to examine OTA adsorption by two strains of Saccharomyces cerevisiae (the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a yeast cell wall preparation. Experiments were conducted using Nero di Troia red wine contaminated with 2 μg/L OTA and supplemented with yeast biomass (20 g/L). The samples were analyzed periodically to assess mycotoxin concentration, chromatic characteristics, and total anthocyanins over 84 days of aging. Yeast cell walls revealed the highest OTA-adsorption in comparison to thermally-inactivated cells (50% vs. 43% toxin reduction), whilst no significant differences were found for the amount of adsorbed anthocyanins in OTA-contaminated and control wines. OTA and anthocyanins adsorption were not competitive phenomena. Unfortunately, the addition of yeast cells to wine could cause color loss; therefore, yeast selection should also focus on this trait to select the best strain. PMID:26516913

  8. Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

    NASA Astrophysics Data System (ADS)

    Chen, Yanjie; Zhang, Quanqi; Qi, Jie; Sun, Yeying; Zhong, Qiwang; Wang, Xubo; Wang, Zhigang; Li, Shuo; Li, Chunmei

    2009-02-01

    Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

  9. 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...

  10. Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Datta, A.R.; Wentz, B.A.; Hill, W.E.

    1987-09-01

    A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.

  11. Human parainfluenza virus type 2 V protein inhibits caspase-1.

    PubMed

    Ohta, Keisuke; Matsumoto, Yusuke; Nishio, Machiko

    2018-04-01

    The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1β (IL-1β) in a dose-dependent manner. IL-1β secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1β via its interaction with caspase-1.

  12. A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

    PubMed Central

    2013-01-01

    Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has

  13. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

    PubMed Central

    Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Qin, Jiufu; Siewers, Verena; Nielsen, Jens

    2016-01-01

    Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value. PMID:27222209

  14. Electronic Properties, Screening, and Efficient Carrier Transport in NaSbS 2

    DOE PAGES

    Sun, Jifeng; Singh, David J.

    2017-02-13

    NaSbS 2 is a semiconductor that was recently shown to have remarkable efficacy as a solar absorber indicating efficient charge collection even in material containing defects. We report first-principles calculations of properties that show (1) an indirect gap only slightly smaller than the direct gap, which may impede the recombination of photoexcited carriers, (2) highly anisotropic electronic and optical properties reflecting a layered crystal structure, (3) a pushed-up valence-band maximum due to repulsion from the Sb 5s states, and (4) cross-gap hybridization between the S p—derived valence bands and the Sb 5p states. This latter feature leads to enhanced Bornmore » effective charges that can provide local screening and, therefore, defect tolerance. Finally, these features are discussed in relation to the performance of the compound as a semiconductor with efficient charge collection.« less

  15. Effect of wine yeast monoculture practice on the biodiversity of non-Saccharomyces yeasts.

    PubMed

    Ganga, M A; Martínez, C

    2004-01-01

    The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non-Saccharomyces yeasts in wine-producing areas in Chile. Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of beta-glycosidase. The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non-Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions.

  16. Big data mining powers fungal research: recent advances in fission yeast systems biology approaches.

    PubMed

    Wang, Zhe

    2017-06-01

    Biology research has entered into big data era. Systems biology approaches therefore become the powerful tools to obtain the whole landscape of how cell separate, grow, and resist the stresses. Fission yeast Schizosaccharomyces pombe is wonderful unicellular eukaryote model, especially studying its division and metabolism can facilitate to understanding the molecular mechanism of cancer and discovering anticancer agents. In this perspective, we discuss the recent advanced fission yeast systems biology tools, mainly focus on metabolomics profiling and metabolic modeling, protein-protein interactome and genetic interaction network, DNA sequencing and applications, and high-throughput phenotypic screening. We therefore hope this review can be useful for interested fungal researchers as well as bioformaticians.

  17. Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?

    PubMed Central

    Saxena, Shailaja Gada; Desai, Kundanbala; Shewale, Lata; Ranjan, Prabhat

    2014-01-01

    CONTEXT: There is a high incidence of numerical chromosomal aberration in couples with repeated in vitro fertilization (IVF) failure, advanced maternal age, repeated unexplained abortions, severe male factor infertility and unexplained infertility. Pre-implantation genetic screening (PGS), a variant of pre-implantation genetic diagnosis, screens numerical chromosomal aberrations in couples with normal karyotype, experiencing poor reproductive outcome. The present study includes the results of the initial pilot study on 9 couples who underwent 10 PGS cycles. AIM: The aim of the present study was to evaluate the beneficial effects of PGS in couples with poor reproductive outcome. SETTINGS AND DESIGN: Data of initial 9 couples who underwent 10 PGS for various indications was evaluated. SUBJECTS AND METHODS: Blastomere biopsy was performed on cleavage stage embryos and subjected to two round fluorescence in situ hybridization (FISH) testing for chromosomes 13, 18, 21, X and Y as a two-step procedure. RESULTS: Six of the 9 couples (10 PGS cycles) conceived, including a twin pregnancy in a couple with male factor infertility, singleton pregnancies in a couple with secondary infertility, in three couples with adverse obstetric outcome in earlier pregnancies and in one couple with repeated IVF failure. CONCLUSION: In the absence of availability of array-comparative genomic hybridization in diagnostic clinical scenario for PGS and promising results with FISH based PGS as evident from the current pilot study, it is imperative to offer the best available services in the present scenario for better pregnancy outcome for patients. PMID:24829527

  18. 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...

  19. Clinical Validation of Anyplex II HPV HR Detection Test for Cervical Cancer Screening in Korea.

    PubMed

    Jung, Sunkyung; Lee, Byungdoo; Lee, Kap No; Kim, Yonggoo; Oh, Eun-Jee

    2016-03-01

    The Anyplex II HPV HR detection kit (Seegene Inc, Seoul, Korea) is a new, multiplex, real-time polymerase chain reaction assay to detect individual 14 high-risk (HR) human papillomavirus (HPV) types in a single tube. To evaluate the clinical performance of the HPV HR kit in predicting high-grade squamous intraepithelial lesions and cervical intraepithelial lesions grade 2 or worse in cervical cancer screening. We analyzed 1137 cervical samples in Huro Path medium (CelltraZone, Seoul, Korea) from Korean women. The clinical performance of the HPV HR kit was compared with Hybrid Capture 2 (Qiagen, Valencia, California) using the noninferiority score test in a routine cervical cancer screening setting. The intralaboratory and interlaboratory agreements of HPV HR were also evaluated. Overall agreement between the 2 assays was 92.4% (1051 of 1137) with a κ value of 0.787. Clinical sensitivity of HPV HR for high-grade squamous intraepithelial lesions and cervical intraepithelial lesions grade 2 or worse was 94.4% (95% confidence interval [CI], 89.2-99.7) and 92.5% (95% CI, 84.3-100.0), respectively. The respective values for Hybrid Capture 2 were 93.1% (95% CI, 87.2-98.9) and 87.5% (95% CI, 77.3-99.7). Clinical sensitivity and specificity of HPV HR were not inferior to those of Hybrid Capture 2 (P = .005 and P = .04, respectively). The HPV HR showed good intralaboratory and interlaboratory reproducibility at 98.0% (κ = 0.953) and 97.4% (κ = 0.940), respectively. The HPV HR demonstrates comparable performance to the Hybrid Capture 2 test and can be useful for HPV-based cervical cancer screening testing.

  20. Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies

    PubMed Central

    Lasserre, Jean-Paul; Dautant, Alain; Aiyar, Raeka S.; Kucharczyk, Roza; Glatigny, Annie; Tribouillard-Tanvier, Déborah; Rytka, Joanna; Blondel, Marc; Skoczen, Natalia; Reynier, Pascal; Pitayu, Laras; Rötig, Agnès; Delahodde, Agnès; Steinmetz, Lars M.; Dujardin, Geneviève; Procaccio, Vincent; di Rago, Jean-Paul

    2015-01-01

    ABSTRACT Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast. PMID:26035862