Sample records for yeast screening system

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. Yeast killer systems.

    PubMed Central

    Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

    1997-01-01

    The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. Evaluation of Automated Yeast Identification System

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.

    1996-01-01

    One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions.

    PubMed

    Fukuda, Nobuo; Ishii, Jun; Kondo, Akihiko

    2011-09-01

    Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gγ recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gγ recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 × 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wild-type haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification. © 2011 The Authors Journal compilation © 2011 FEBS.

  10. 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

  11. 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.

  12. Experimental Systems to Study Yeast Pexophagy.

    PubMed

    Yamashita, Shun-Ichi; Oku, Masahide; Sakai, Yasuyoshi; Fujiki, Yukio

    2017-01-01

    Peroxisome abundance is tightly regulated according to the physiological contexts, through regulations of both proliferation and degradation of the organelles. Here, we describe detailed methods to analyze processes for autophagic degradation of peroxisomes, termed pexophagy, in yeast organisms. The assay systems include a method for biochemical detection of pexophagy completion, and one for microscopic visualization of specialized membrane structures acting in pexophagy. As a model yeast organism utilized in studies of pexophagy, the methylotrophic yeast Komagataella phaffii (Pichia pastoris) is referred to in this chapter and related information on the studies with baker's yeast (Saccharomyces cerevisiae) is also included. The described techniques facilitate elucidation of molecular machineries for pexophagy and understanding of peroxisome-selective autophagic pathways.

  13. 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.

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

    PubMed Central

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

    2016-01-01

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

  15. [Treatment of oil-manufacturing wastewater by yeast-SBR system].

    PubMed

    Lü, Wen-zhou; Liu, Ying; Huang, Yi-zhen

    2008-04-01

    Eight yeast strains were applied to a sequencing batch reactor (SBR) to treat high-strength oil-containing wastewater. The removal performance, yeast cultivation method and key factors affecting the stability of system were discussed. The results show yeast sludge with MLSS of 19 g/L and SVI of 35 mL/g can be obtained in 6 d in an open system without any molds and bacteria inhibitor addition; In 30 d continuous wastewater treatment, COD and oil removal rate achieve 86.8%-96.9% and above 99.5% respectively under the influent conditions of the COD of 9000-23000 mg/L and oil of 4500-16000 mg/L; Short period of pH impact brings reversible effects on the system and the sludge retention time can affect the SVI of the yeast; Absence of nitrogen induces morphology conversion of some yeast cells from single cell to filamentous one and impairs the settling capability of the yeast.

  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. 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.

  18. Identifying genes that extend life span using a high-throughput screening system.

    PubMed

    Chen, Cuiying; Contreras, Roland

    2007-01-01

    We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

  19. Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system.

    PubMed

    Wolf, Sylvi; Diel, Patrick; Parr, Maria Kristina; Rataj, Felicitas; Schänzer, Willhelm; Vollmer, Günter; Zierau, Oliver

    2011-04-01

    The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0-307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

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

    PubMed

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

    2012-02-01

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

  6. Effects of cryoprotectants on the viability and activity of freeze dried recombinant yeasts as novel oral drug delivery systems assessed by an artificial digestive system.

    PubMed

    Blanquet, Stéphanie; Garrait, Ghislain; Beyssac, Erick; Perrier, Céline; Denis, Sylvain; Hébrard, Géraldine; Alric, Monique

    2005-09-01

    The aim of this study was to investigate, in a gastric-small intestinal system TIM-1, the effect of cryoprotectants on the survival of freeze-dried Saccharomyces cerevisiae expressing the heterologous P450 73A1 and their ability to convert trans-cinnamic acid into p-coumaric acid. Yeasts were lyophilized in suspensions of trehalose, maltose, lactose, or a milk proteins/trehalose mix. Freeze-dried or native yeasts and trans-cinnamic acid were introduced simultaneously into TIM-1 at the beginning of digestion. Yeast survival rate was evaluated by cell counting in the ileal effluents. P450 73A1 activity was followed by HPLC assay of p-coumaric acid. Freeze-dried yeasts showed high tolerance to digestive conditions. Nevertheless, their survival rate was lower than that of non-dried cells (around 80% whatever the protective agent vs. 96%). The ability of recombinant freeze-dried S. cerevisiae to perform a bioconversion reaction in the digestive tract was shown with all the protectants. The highest trans-cinnamic acid conversion rate (24 vs. 41% for native yeasts) was obtained with the milk proteins/trehalose mix. These results show that freeze-drying might be considered for the pharmaceutical formulation of new drug delivery systems based on orally administered recombinant yeasts and that TIM-1 could be a helpful tool for the pre-screening of oral dosage forms.

  7. 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.

  8. 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.

  9. 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

  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. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. Evaluation of DuPont Qualicon Bax System PCR assay for yeast and mold.

    PubMed

    Wallace, F Morgan; Burns, Frank; Fleck, Lois; Andaloro, Bridget; Farnum, Andrew; Tice, George; Ruebl, Joanne

    2010-01-01

    Evaluations were conducted to test the performance of the BAX System PCR assay which was certified as Performance Tested Method 010902 for screening yeast and mold in yogurt, corn starch, and milk-based powdered infant formula. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent to the U.S. Food and Drug Administration's Bacteriological Analytical Manual culture method, but with a significantly shorter time to obtain results. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined affected the performance of the assay.

  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. Comparison of Three Commercial Systems for Identification of Yeasts Commonly Isolated in the Clinical Microbiology Laboratory

    PubMed Central

    Wadlin, Jill K.; Hanko, Gayle; Stewart, Rebecca; Pape, John; Nachamkin, Irving

    1999-01-01

    We evaluated three commercial systems (RapID Yeast Plus System; Innovative Diagnostic Systems, Norcross, Ga.; API 20C Aux; bioMerieux-Vitek, Hazelwood, Mo.; and Vitek Yeast Biochemical Card, bioMerieux-Vitek) against an auxinographic and microscopic morphologic reference method for the ability to identify yeasts commonly isolated in our clinical microbiology laboratory. Two-hundred one yeast isolates were compared in the study. The RapID Yeast Plus System was significantly better than either API 20C Aux (193 versus 167 correct identifications; P < 0.0001) or the Vitek Yeast Biochemical Card (193 versus 173 correct identifications; P = 0.003) for obtaining correct identifications to the species level without additional testing. There was no significant difference between results obtained with API 20C Aux and the Vitek Yeast Biochemical Card system (P = 0.39). The API 20C Aux system did not correctly identify any of the Candida krusei isolates (n = 23) without supplemental testing and accounted for the major differences between the API 20C Aux and RapID Yeast Plus systems. Overall, the RapID Yeast Plus System was easy to use and is a good system for the routine identification of clinically relevant yeasts. PMID:10325356

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Yeast Immobilization Systems for Alcoholic Wine Fermentations: Actual Trends and Future Perspectives

    PubMed Central

    Moreno-García, Jaime; García-Martínez, Teresa; Mauricio, Juan C.; Moreno, Juan

    2018-01-01

    Yeast immobilization is defined as the physical confinement of intact cells to a region of space with conservation of biological activity. The use of these methodologies for alcoholic fermentation (AF) offers many advantages over the use of the conventional free yeast cell method and different immobilization systems have been proposed so far for different applications, like winemaking. The most studied methods for yeast immobilization include the use of natural supports (e.g., fruit pieces), organic supports (e.g., alginate), inorganic (e.g., porous ceramics), membrane systems, and multi-functional agents. Some advantages of the yeast-immobilization systems include: high cell densities, product yield improvement, lowered risk of microbial contamination, better control and reproducibility of the processes, as well as reuse of the immobilization system for batch fermentations and continuous fermentation technologies. However, these methods have some consequences on the behavior of the yeasts, affecting the final products of the fermentative metabolism. This review compiles current information about cell immobilizer requirements for winemaking purposes, the immobilization methods applied to the production of fermented beverages to date, and yeast physiological consequences of immobilization strategies. Finally, a recent inter-species immobilization methodology has been revised, where yeast cells are attached to the hyphae of a Generally Recognized As Safe fungus and remain adhered following loss of viability of the fungus. The bio-capsules formed with this method open new and promising strategies for alcoholic beverage production (wine and low ethanol content beverages). PMID:29497415

  7. 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

  8. Yeast Prions: Structure, Biology, and Prion-Handling Systems

    PubMed Central

    Shewmaker, Frank P.; Bateman, David A.; Edskes, Herman K.; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E.

    2015-01-01

    SUMMARY A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. PMID:25631286

  9. 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.

  10. 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

  11. Yeast prions: structure, biology, and prion-handling systems.

    PubMed

    Wickner, Reed B; Shewmaker, Frank P; Bateman, David A; Edskes, Herman K; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E

    2015-03-01

    A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. CRISPR/Cas system for yeast genome engineering: advances and applications

    PubMed Central

    Stovicek, Vratislav; Holkenbrink, Carina

    2017-01-01

    Abstract The methods based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system have quickly gained popularity for genome editing and transcriptional regulation in many organisms, including yeast. This review aims to provide a comprehensive overview of CRISPR application for different yeast species: from basic principles and genetic design to applications. PMID:28505256

  13. 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...

  14. 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.

  15. A systems-level approach for metabolic engineering of yeast cell factories.

    PubMed

    Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

    2012-03-01

    The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. 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...

  17. 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

  18. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    PubMed

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B

    2014-05-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  19. Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

    PubMed Central

    Duina, Andrea A.; Miller, Mary E.; Keeney, Jill B.

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans. PMID:24807111

  20. 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.

  1. 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.

  2. Use of one- and two-mediator systems for developing a BOD biosensor based on the yeast Debaryomyces hansenii.

    PubMed

    Zaitseva, A S; Arlyapov, V A; Yudina, N Yu; Alferov, S V; Reshetilov, A N

    2017-03-01

    We investigated the use of one- and two-mediator systems in amperometric BOD biosensors (BOD, biochemical oxygen demand) based on the yeast Debaryomyces hansenii. Screening of nine mediators potentially capable of electron transfer - ferrocene, 1,1'-dimethylferrocene, ferrocenecarboxaldehyde, ferroceneacetonitrile, neutral red, 2,6-dichlorophenolindophenol, thionine, methylene blue and potassium ferricyanide - showed only ferrocene and neutral red to be efficient electron carriers for the eukaryotes studied. Two-mediator systems based on combinations of the investigated compounds were used to increase the efficiency of electron transfer. The developed two-mediator biosensors exceeded their one-mediator analogs by their characteristics. The most preferable two-mediator system for developing a BOD biosensor was a ferrocene-methylene blue combination that ensured a satisfactory long-time stability (43 days), selectivity, sensitivity (the lower limit of the determined BOD 5 concentrations, 2.5mg О 2 /dm 3 ) and speed (assay time for one sample, not greater than 10min) of BOD determination. Analysis of water samples showed that the use of a ferrocene-methylene blue two-mediator system and the yeast D. hansenii enabled registration of data that highly correlated with the results of the standard method (R=0.9913). Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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.

  4. Assessment of Multi Fragment Melting Analysis System (MFMAS) for the Identification of Food-Borne Yeasts.

    PubMed

    Kesmen, Zülal; Büyükkiraz, Mine E; Özbekar, Esra; Çelik, Mete; Özkök, F Özge; Kılıç, Özge; Çetin, Bülent; Yetim, Hasan

    2018-06-01

    Multi Fragment Melting Analysis System (MFMAS) is a novel approach that was developed for the species-level identification of microorganisms. It is a software-assisted system that performs concurrent melting analysis of 8 different DNA fragments to obtain a fingerprint of each strain analyzed. The identification is performed according to the comparison of these fingerprints with the fingerprints of known yeast species recorded in a database to obtain the best possible match. In this study, applicability of the yeast version of the MFMAS (MFMAS-yeast) was evaluated for the identification of food-associated yeast species. For this purpose, in this study, a total of 145 yeast strains originated from foods and beverages and 19 standard yeast strains were tested. The DNAs isolated from these yeast strains were analyzed by the MFMAS, and their species were successfully identified with a similarity rate of 95% or higher. It was shown that the strains belonged to 43 different yeast species that are widely found in the foods. A clear discrimination was also observed in the phylogenetically related species. In conclusion, it might be suggested that the MFMAS-yeast seems to be a highly promising approach for a rapid, accurate, and one-step identification of the yeasts isolated from food products and/or their processing environments.

  5. 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

  6. 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.

  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. 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

  9. A multiplex culture system for the long-term growth of fission yeast cells.

    PubMed

    Callens, Céline; Coelho, Nelson C; Miller, Aaron W; Sananes, Maria Rosa Domingo; Dunham, Maitreya J; Denoual, Matthieu; Coudreuse, Damien

    2017-08-01

    Maintenance of long-term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin-encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo-pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long-term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

  10. 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

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

    PubMed

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

    2011-09-01

    The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

    DOE PAGES

    Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; ...

    2018-02-20

    The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

  20. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

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

    Lian, Jiazhang; Bao, Zehua; Hu, Sumeng

    The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. Furthermore, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to constructmore » xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.« less

  1. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

    PubMed

    Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

    2018-06-01

    The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.

  2. 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

  3. Evaluation of the Microbial Identification System for identification of clinically isolated yeasts.

    PubMed Central

    Crist, A E; Johnson, L M; Burke, P J

    1996-01-01

    The Microbial Identification System (MIS; Microbial ID, Inc., Newark, Del.) was evaluated for the identification of 550 clinically isolated yeasts. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C and conventional methods) and included Candida albicans (n = 294), C. glabrata (n = 145), C. tropicalis (n = 58), C. parapsilosis (n = 33), and other yeasts (n = 20). In preparation for fatty acid analysis, yeasts were inoculated onto Sabouraud dextrose agar and incubated at 28 degrees C for 24 h. Yeasts were harvested, saponified, derivatized, and extracted, and fatty acid analysis was performed according to the manufacturer's instructions. Fatty acid profiles were analyzed, and computer identifications were made with the Yeast Clinical Library (database version 3.8). Of the 550 isolates tested, 374 (68.0%) were correctly identified to the species level, with 87 (15.8%) being incorrectly identified and 89 (16.2%) giving no identification. Repeat testing of isolates giving no identification resulted in an additional 18 isolates being correctly identified. This gave the MIS an overall identification rate of 71.3%. The most frequently misidentified yeast was C. glabrata, which was identified as Saccharomyces cerevisiae 32.4% of the time. On the basis of these results, the MIS, with its current database, does not appear suitable for the routine identification of clinically important yeasts. PMID:8880489

  4. A multiplex culture system for the long‐term growth of fission yeast cells

    PubMed Central

    Callens, Céline; Coelho, Nelson C.; Miller, Aaron W.; Sananes, Maria Rosa Domingo; Dunham, Maitreya J.; Denoual, Matthieu

    2017-01-01

    Abstract Maintenance of long‐term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin‐encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo‐pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long‐term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:28426144

  5. Evaluation of the Biolog MicroStation system for yeast identification

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.; Molina, T. C.; Pierson, D. L.; Mishra, S. K.

    1996-01-01

    One hundred and fifty-nine isolates representing 16 genera and 53 species of yeasts were processed with the Biolog MicroStation System for yeast identification. Thirteen genera and 38 species were included in the Biolog database. For these 129 isolates, correct identifications to the species level were 13.2, 39.5 and 48.8% after 24, 48 and 72 hours incubation at 30 degrees C, respectively. Three genera and 15 species which were not included in the Biolog database were also tested. Of the 30 isolates studied, 16.7, 53.3 and 56.7% of the isolates were given incorrect names from the system's database after 24,48 and 72 h incubation at 30 degrees C, respectively. The remaining isolates of this group were not identified.

  6. 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

  7. 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.

  8. Development of an integrated electrochemical system for in vitro yeast viability testing.

    PubMed

    Adami, Andrea; Ress, Cristina; Collini, Cristian; Pedrotti, Severino; Lorenzelli, Leandro

    2013-02-15

    This work describes the development and testing of a microfabricated sensor for rapid cell growth monitoring, especially focused on yeast quality assessment for wine applications. The device consists of a NMOS ISFET sensor with Si(3)N(4) gate, able to indirectly monitor extracellular metabolism through pH variation of the medium, and a solid-state reference electrode implemented with PVC membranes doped with lipophilic salts (tetrabutylammonium-tetrabutylborate (TBA-TBB) and Potassium tetrakis(4-chlorphenyl)borate (KTClpB)). The use of a solid state reference electrode enables the implementation of a large number of cell assays in parallel, without the need of external conventional reference electrodes. Microbial growth testing has been performed both in standard culture conditions and on chip at different concentrations of ethanol in order to carry out a commonly used screening of wine yeast strains. Cell growth tests can be performed in few hours, providing a fast, sensitive and low cost analysis with respect to the conventional procedures. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. 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.

  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. 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

  12. 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.

  13. [Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

    PubMed

    Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B

    2016-01-01

    For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

  14. 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

  15. Solving the puzzle of yeast survival in ephemeral nectar systems: exponential growth is not enough.

    PubMed

    Hausmann, Sebastian L; Tietjen, Britta; Rillig, Matthias C

    2017-12-01

    Flower nectar is a sugar-rich ephemeral habitat for microorganisms. Nectar-borne yeasts are part of the microbial community and can affect pollination by changing nectar chemistry, attractiveness to pollinators or flower temperature if yeast population densities are high. Pollinators act as dispersal agents in this system; however, pollination events lead potentially to shrinking nectar yeast populations. We here examine how sufficiently high cell densities of nectar yeast can develop in a flower. In laboratory experiments, we determined the remaining fraction of nectar yeast cells after nectar removal, and used honeybees to determine the number of transmitted yeast cells from one flower to the next. The results of these experiments directly fed into a simulation model providing an insight into movement and colonization ecology of nectar yeasts. We found that cell densities only reached an ecologically relevant size for an intermediate pollination probability. Too few pollination events reduce yeast inoculation rate and too many reduce yeast population size strongly. In addition, nectar yeasts need a trait combination of at least an intermediate growth rate and an intermediate remaining fraction to compensate for highly frequent decimations. Our results can be used to predict nectar yeast dispersal, growth and consequently their ecological effects. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology.

    PubMed

    Wickner, R B; Edskes, H K; Gorkovskiy, A; Bezsonov, E E; Stroobant, E E

    2016-01-01

    Yeast prions (infectious proteins) were discovered by their outré genetic properties and have become important models for an array of human prion and amyloid diseases. A single prion protein can become any of many distinct amyloid forms (called prion variants or strains), each of which is self-propagating, but with different biological properties (eg, lethal vs mild). The folded in-register parallel β sheet architecture of the yeast prion amyloids naturally suggests a mechanism by which prion variant information can be faithfully transmitted for many generations. The yeast prions rely on cellular chaperones for their propagation, but can be cured by various chaperone imbalances. The Btn2/Cur1 system normally cures most variants of the [URE3] prion that arise. Although most variants of the [PSI+] and [URE3] prions are toxic or lethal, some are mild in their effects. Even the most mild forms of these prions are rare in the wild, indicating that they too are detrimental to yeast. The beneficial [Het-s] prion of Podospora anserina poses an important contrast in its structure, biology, and evolution to the yeast prions characterized thus far. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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

  18. 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

  19. 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.

  20. Yeast cell surface display for lipase whole cell catalyst and its applications

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

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

  1. 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

  2. Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

    PubMed

    Andreu, Cecilia; Del Olmo, Marcel Lí

    2018-03-01

    Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

  3. 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.

  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. 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.

  6. 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

  7. 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

  8. 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.

  9. Limitations of the Current Microbial Identification System for Identification of Clinical Yeast Isolates

    PubMed Central

    Kellogg, James A.; Bankert, David A.; Chaturvedi, Vishnu

    1998-01-01

    The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28°C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer’s instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as “no match” by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of Candida glabrata, the predictive value of an MIS identification of unknown isolates as C. glabrata was 100% (102 of 102) because no isolates of other species were misidentified as C. glabrata. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of Saccharomyces cerevisiae, the predictive value of an MIS identification of unknown isolates as S. cerevisiae was only 47% (15 of 32), because 17 isolates of C. glabrata were misidentified as S. cerevisiae. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved. PMID:9574676

  10. 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.

  11. 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.

  12. Saccharomyces cerevisiae Produces a Yeast Substance that Exhibits Estrogenic Activity in Mammalian Systems

    NASA Astrophysics Data System (ADS)

    Feldman, David; Stathis, Peter A.; Hirst, Margaret A.; Price Stover, E.; Do, Yung S.; Kurz, Walter

    1984-06-01

    Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

  13. 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.

  14. 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

  15. 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.

  16. New features of mitochondrial DNA replication system in yeast and man.

    PubMed

    Lecrenier, N; Foury, F

    2000-04-04

    In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

  17. 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

  18. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast

    PubMed Central

    Pang, Wei; Coghill, George M.

    2015-01-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. PMID:25864377

  19. 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.

  20. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

    USDA-ARS?s Scientific Manuscript database

    A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

  1. Yeast cell differentiation: Lessons from pathogenic and non-pathogenic yeasts.

    PubMed

    Palková, Zdena; Váchová, Libuše

    2016-09-01

    Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

    PubMed Central

    Westblade, Lars F.; Jennemann, Rebecca; Branda, John A.; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B.; Ginocchio, Christine C.; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Rychert, Jenna A.; Sercia, Linda

    2013-01-01

    The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories. PMID:23658267

  3. Multicenter study evaluating the Vitek MS system for identification of medically important yeasts.

    PubMed

    Westblade, Lars F; Jennemann, Rebecca; Branda, John A; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B; Ginocchio, Christine C; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Rychert, Jenna A; Sercia, Linda; Burnham, Carey-Ann D

    2013-07-01

    The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

  4. Beer and bread to brains and beyond: can yeast cells teach us about neurodegenerative disease?

    PubMed

    Gitler, Aaron D

    2008-01-01

    For millennia, humans have harnessed the astonishing power of yeast, producing such culinary masterpieces as bread, beer and wine. Therefore, in this new millennium, is it very farfetched to ask if we can also use yeast to unlock some of the modern day mysteries of human disease? Remarkably, these seemingly simple cells possess most of the same basic cellular machinery as the neurons in the brain. We and others have been using the baker's yeast, Saccharomyces cerevisiae, as a model system to study the mechanisms of devastating neurodegenerative diseases such as Parkinson's, Huntington's, Alzheimer's and amyotrophic lateral sclerosis. While very different in their pathophysiology, they are collectively referred to as protein-misfolding disorders because of the presence of misfolded and aggregated forms of various proteins in the brains of affected individuals. Using yeast genetics and the latest high-throughput screening technologies, we have identified some of the potential causes underpinning these disorders and discovered conserved genes that have proven effective in preventing neuron loss in animal models. Thus, these genes represent new potential drug targets. In this review, I highlight recent work investigating mechanisms of cellular toxicity in a yeast Parkinson's disease model and discuss how similar approaches are being applied to additional neurodegenerative diseases.

  5. 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.

  6. Yeast Genetics and Biotechnological Applications

    NASA Astrophysics Data System (ADS)

    Mishra, Saroj; Baranwal, Richa

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

  7. 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.

  8. Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

    PubMed

    Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

    2006-04-28

    Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

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

    PubMed

    Song, M; Ouyang, Z; Liu, Z L

    2009-05-01

    Composed of linear difference equations, a discrete dynamical system (DDS) model was designed to reconstruct transcriptional regulations in gene regulatory networks (GRNs) for ethanologenic yeast Saccharomyces cerevisiae in response to 5-hydroxymethylfurfural (HMF), a bioethanol conversion inhibitor. The modelling aims at identification of a system of linear difference equations to represent temporal interactions among significantly expressed genes. Power stability is imposed on a system model under the normal condition in the absence of the inhibitor. Non-uniform sampling, typical in a time-course experimental design, is addressed by a log-time domain interpolation. A statistically significant DDS model of the yeast GRN derived from time-course gene expression measurements by exposure to HMF, revealed several verified transcriptional regulation events. These events implicate Yap1 and Pdr3, transcription factors consistently known for their regulatory roles by other studies or postulated by independent sequence motif analysis, suggesting their involvement in yeast tolerance and detoxification of the inhibitor.

  10. 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.

  11. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

    PubMed

    Pang, Wei; Coghill, George M

    2015-05-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  12. 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

  13. 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

  14. Genomics and the making of yeast biodiversity

    USDA-ARS?s Scientific Manuscript database

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces ...

  15. A systems-biology approach to yeast actin cables.

    PubMed

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2012-01-01

    We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin-cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin-monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions - among actin-monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins - and the emergence of cell-scale physical form as embodied by the actin cables themselves.

  16. (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.

  17. (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

  18. 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

  19. 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

  20. 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.

  1. 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®

  2. Variation in Microbial Identification System Accuracy for Yeast Identification Depending on Commercial Source of Sabouraud Dextrose Agar

    PubMed Central

    Kellogg, James A.; Bankert, David A.; Chaturvedi, Vishnu

    1999-01-01

    The accuracy of the Microbial Identification System (MIS; MIDI, Inc.) for identification of yeasts to the species level was compared by using 438 isolates grown on prepoured BBL Sabouraud dextrose agar (SDA) and prepoured Remel SDA. Correct identification was observed for 326 (74%) of the yeasts cultured on BBL SDA versus only 214 (49%) of yeasts grown on Remel SDA (P < 0.001). The commercial source of the SDA used in the MIS procedure significantly influences the system’s accuracy. PMID:10325387

  3. A Systems-Biology Approach to Yeast Actin Cables

    PubMed Central

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2011-01-01

    We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions—among actin monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins—and the emergence of cell-scale physical form as embodied by the actin cables themselves. PMID:22161338

  4. 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.

  5. 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...

  6. 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

  7. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

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

  8. Cellodextrin transport in yeast for improved biofuel production.

    PubMed

    Galazka, Jonathan M; Tian, Chaoguang; Beeson, William T; Martinez, Bruno; Glass, N Louise; Cate, Jamie H D

    2010-10-01

    Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes efficient growth of this yeast on cellodextrins. In simultaneous saccharification and fermentation experiments, the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system.

  9. 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

  10. 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

  11. [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.

  12. 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.

  13. Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing.

    PubMed

    Pfannebecker, Jens; Schiffer-Hetz, Claudia; Fröhlich, Jürgen; Becker, Barbara

    2016-11-01

    In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (a w )-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS ® parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1-0.2% ammonium salts ((NH 4 ) 2 HPO 4 , (NH 4 ) 2 SO 4 or NH 4 NO 3 ), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 10 2 CFU/g within a time period of 2-3days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124h in YEG50, 95.5h in MYG50 and 55h in OM bouillon. Compared to YEG50 the maximum CO 2 transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3days for Z. rouxii and T. delbrueckii

  14. Yeast: A Research Organism for Teaching Genetics.

    ERIC Educational Resources Information Center

    Manney, Thomas R.; Manney, Monta L.

    1992-01-01

    Explains why laboratory strains of bakers yeast, Saccharomyces cerevisiae, are particularly suited for classroom science activities. Describes the sexual life cycle of yeast and the genetic system with visible mutations. Presents an overview of activities that can be done with yeast and gives a source for teachers to obtain more information. (PR)

  15. 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

  16. 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.

  17. The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

    PubMed

    Siggers, Keri A; Lesser, Cammie F

    2008-07-17

    Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

  18. Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinically Important Bacteria and Yeasts.

    PubMed

    Wilson, Deborah A; Young, Stephen; Timm, Karen; Novak-Weekley, Susan; Marlowe, Elizabeth M; Madisen, Neil; Lillie, Jennifer L; Ledeboer, Nathan A; Smith, Rebecca; Hyke, Josh; Griego-Fullbright, Christen; Jim, Patricia; Granato, Paul A; Faron, Matthew L; Cumpio, Joven; Buchan, Blake W; Procop, Gary W

    2017-06-01

    A report on the multicenter evaluation of the Bruker MALDI Biotyper CA System (MBT-CA; Bruker Daltonics, Billerica, MA) for the identification of clinically important bacteria and yeasts. In total, 4,399 isolates of medically important bacteria and yeasts were assessed in the MBT-CA. These included 2,262 aerobic gram-positive (AGP) bacteria, 792 aerobic gram-negative (AGN) bacteria 530 anaerobic (AnA) bacteria, and 815 yeasts (YSTs). Three processing methods were assesed. Overall, 98.4% (4,329/4,399) of all bacterial and yeast isolates were correctly identified to the genus and species/species complex level, and 95.7% of isolates were identified with a high degree of confidence. The percentage correctly identified and the percentage identified correctly with a high level of confidence, respectively, were as follows: AGP bacteria (98.6%/96.5%), AGN bacteria (98.5%/96.8%), AnA bacteria (98.5%/97.4%), and YSTs (97.8%/87.6%). The extended direct transfer method was only minimally superior to the direct transfer method for bacteria (89.9% vs 86.8%, respectively) but significantly superior for yeast isolates (74.0% vs 48.9%, respectively). The Bruker MALDI Biotyper CA System accurately identifies most clinically important bacteria and yeasts and has optional processing methods to improve isolate characterization. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  19. 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.

  20. Screening the yeast genome for energetic metabolism pathways involved in a phenotypic response to the anti-cancer agent 3-bromopyruvate

    PubMed Central

    Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H.; Pedersen, Peter L.; Goffeau, Andre; Ułaszewski, Stanisław

    2016-01-01

    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. PMID:26862728

  1. Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast

    PubMed Central

    2011-01-01

    Background The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. Results To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. Conclusions As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs. PMID:22023736

  2. 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

  3. 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

  4. 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.

  5. [Distiller Yeasts Producing Antibacterial Peptides].

    PubMed

    Klyachko, E V; Morozkina, E V; Zaitchik, B Ts; Benevolensky, S V

    2015-01-01

    A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

  6. [Evaluation of common commercial systems for the identification of yeast isolates in microbiology laboratories: a multicenter study].

    PubMed

    Karabıçak, Nilgün; Uludağ Altun, Hatice; Karatuna, Onur; Hazırolan, Gülşen; Aksu, Neriman; Adiloğlu, Ali; Akyar, Işın

    2015-04-01

    Accurate and rapid identification of yeast isolates have become important in recent years for not only antifungal susceptibility testing due to the species-specific clinical resistance breakpoints but also early initiation of appropriate antifungal therapy. In clinical microbiology laboratories species identification of yeasts is often performed with several commercial systems based on biochemical properties and rarely according to the physiological and morphological characteristics. The aim of this study was to compare the two common commercial systems, VITEK 2 YST ID Card (Vitek; bioMérieux, France) and API 20C AUX (API; bioMérieux, France) with conventional mycological methods. A total of 473 clinical yeast strains isolated from clinical specimens in different university and training/research hospitals and identified by Vitek system were included in the study. The isolates were re-identified with API and conventional methods including morphological identification in the Mycology Reference Laboratory of the Public Health Institute of Turkey. Candida dubliniensis MYA 583, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32268 were used as quality control strains and those standard strains were studied consecutively 10 days with both of the methods. The results of identification by Vitek and API were compared with the results of conventional methods for those 473 yeast isolates [6 genus (Candida, Cryptococcus, Blastoshizomyces, Rhodotorula, Saccharomyces, Trichosporon), 17 species (5 common and 12 rarely isolated)]. The performances of the systems were better (Vitek: 95%; API: 96%) for the commonly detected species (C.albicans, C.parapsilosis, C.glabrata, C.tropicalis and C.krusei) than those for rarely detected species (Vitek: 78.4%; API: 71.6%) (p= 0.155). Misidentification or unidentification were mostly detected for C.parapsilosis (Vitek: 6/87; API: 7/87) and C.glabrata (Vitek: 9/104; API

  7. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

    PubMed Central

    2014-01-01

    Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

  8. 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.

  9. 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.

  10. 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.

  11. Light-mediated control of DNA transcription in yeast

    PubMed Central

    Hughes, Robert M.; Bolger, Steven; Tapadia, Hersh; Tucker, Chandra L.

    2012-01-01

    A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light / cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems. PMID:22922268

  12. Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.

    PubMed

    Smith, Justin D; Suresh, Sundari; Schlecht, Ulrich; Wu, Manhong; Wagih, Omar; Peltz, Gary; Davis, Ronald W; Steinmetz, Lars M; Parts, Leopold; St Onge, Robert P

    2016-03-08

    Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

  13. A general strategy for the evolution of bond-forming enzymes using yeast display

    PubMed Central

    Chen, Irwin; Dorr, Brent M.; Liu, David R.

    2011-01-01

    The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A–catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods. PMID:21697512

  14. Stabilization of the yeast desaturase system by low levels of oxygen

    NASA Technical Reports Server (NTRS)

    Volkmann, C. M.; Klein, H. P.

    1983-01-01

    The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

  15. 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

  16. 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.

  17. 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.

  18. 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 ...

  19. Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

    NASA Astrophysics Data System (ADS)

    Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

    2012-10-01

    In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

  20. Interactions between Drosophila and its natural yeast symbionts—Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

    PubMed Central

    Hoang, Don; Kopp, Artyom

    2015-01-01

    Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker’s yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host–microbe interactions can have profound effects on host biology, the results from D. melanogaster–S. cerevisiae laboratory experiments may not be fully representative of host–microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D

  1. Interactions between Drosophila and its natural yeast symbionts-Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

    PubMed

    Hoang, Don; Kopp, Artyom; Chandler, James Angus

    2015-01-01

    Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker's yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host-microbe interactions can have profound effects on host biology, the results from D. melanogaster-S. cerevisiae laboratory experiments may not be fully representative of host-microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D

  2. Yeast Infection Test

    MedlinePlus

    ... infections of the skin and genitals. Serious yeast infections occur more often in hospital patients and in people with weakened immune systems. References Centers for Disease Control and Prevention [Internet]. Atlanta: U.S. Department of Health ...

  3. Noise screen for attitude control system

    NASA Technical Reports Server (NTRS)

    Rodden, John J. (Inventor); Stevens, Homer D. (Inventor); Hong, David P. (Inventor); Hirschberg, Philip C. (Inventor)

    2002-01-01

    An attitude control system comprising a controller and a noise screen device coupled to the controller. The controller is adapted to control an attitude of a vehicle carrying an actuator system that is adapted to pulse in metered bursts in order to generate a control torque to control the attitude of the vehicle in response to a control pulse. The noise screen device is adapted to generate a noise screen signal in response to the control pulse that is generated when an input attitude error signal exceeds a predetermined deadband attitude level. The noise screen signal comprises a decaying offset signal that when combined with the attitude error input signal results in a net attitude error input signal away from the predetermined deadband level to reduce further control pulse generation.

  4. Genomics and the making of yeast biodiversity.

    PubMed

    Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

    2015-12-01

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. 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.

  6. 14 CFR 29.1105 - Induction system screens.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Powerplant Induction System § 29.1105 Induction system... screen may be in any part of the induction system that is the only passage through which air can reach the engine, unless it can be deiced by heated air; (c) No screen may be deiced by alcohol alone; and...

  7. 14 CFR 25.1105 - Induction system screens.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Powerplant Induction System § 25.1105 Induction system... screen may be in any part of the induction system that is the only passage through which air can reach the engine, unless it can be deiced by heated air; (c) No screen may be deiced by alcohol alone; and...

  8. 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.

  9. Automated Propulsion Data Screening demonstration system

    NASA Technical Reports Server (NTRS)

    Hoyt, W. Andes; Choate, Timothy D.; Whitehead, Bruce A.

    1995-01-01

    A fully-instrumented firing of a propulsion system typically generates a very large quantity of data. In the case of the Space Shuttle Main Engine (SSME), data analysis from ground tests and flights is currently a labor-intensive process. Human experts spend a great deal of time examining the large volume of sensor data generated by each engine firing. These experts look for any anomalies in the data which might indicate engine conditions warranting further investigation. The contract effort was to develop a 'first-cut' screening system for application to SSME engine firings that would identify the relatively small volume of data which is unusual or anomalous in some way. With such a system, limited and expensive human resources could focus on this small volume of unusual data for thorough analysis. The overall project objective was to develop a fully operational Automated Propulsion Data Screening (APDS) system with the capability of detecting significant trends and anomalies in transient and steady-state data. However, the effort limited screening of transient data to ground test data for throttle-down cases typical of the 3-g acceleration, and for engine throttling required to reach the maximum dynamic pressure limits imposed on the Space Shuttle. This APDS is based on neural networks designed to detect anomalies in propulsion system data that are not part of the data used for neural network training. The delivered system allows engineers to build their own screening sets for application to completed or planned firings of the SSME. ERC developers also built some generic screening sets that NASA engineers could apply immediately to their data analysis efforts.

  10. 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.

  11. 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 %).

  12. 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.

  13. Yeast: An Experimental Organism for Modern Biology.

    ERIC Educational Resources Information Center

    Botstein, David; Fink, Gerald R.

    1988-01-01

    Discusses the applicability and advantages of using yeasts as popular and ideal model systems for studying and understanding eukaryotic biology at the cellular and molecular levels. Cites experimental tractability and the cooperative tradition of the research community of yeast biologists as reasons for this success. (RT)

  14. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  15. 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.

  16. 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.

  17. 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

  18. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

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

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodiummore » dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.« less

  19. 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.

  20. 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.

  1. A pathogenesis assay using Saccharomyces cerevisiae and Caenorhabditis elegans reveals novel roles for yeast AP-1, Yap1, and host dual oxidase BLI-3 in fungal pathogenesis.

    PubMed

    Jain, Charu; Yun, Meijiang; Politz, Samuel M; Rao, Reeta Prusty

    2009-08-01

    Treatment of systemic fungal infections is difficult because of the limited number of antimycotic drugs available. Thus, there is an immediate need for simple and innovative systems to assay the contribution of individual genes to fungal pathogenesis. We have developed a pathogenesis assay using Caenorhabditis elegans, an established model host, with Saccharomyces cerevisiae as the invading fungus. We have found that yeast infects nematodes, causing disease and death. Our data indicate that the host produces reactive oxygen species (ROS) in response to fungal infection. Yeast mutants sod1Delta and yap1Delta, which cannot withstand ROS, fail to cause disease, except in bli-3 worms, which carry a mutation in a dual oxidase gene. Chemical inhibition of the NADPH oxidase activity abolishes ROS production in worms exposed to yeast. This pathogenesis assay is useful for conducting systematic, whole-genome screens to identify fungal virulence factors as alternative targets for drug development and exploration of host responses to fungal infections.

  2. 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.

  3. 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

  4. Characteristics of an immobilized yeast cell system using very high gravity for the fermentation of ethanol.

    PubMed

    Ji, Hairui; Yu, Jianliang; Zhang, Xu; Tan, Tianwei

    2012-09-01

    The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280 g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65 h with an average ethanol concentration and ethanol yield of 130.12 g/L and 0.477 g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28 days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75 L. The bioreactor was operated for 26 days at a dilution rate of 0.015 h(-1). The ethanol concentration of the effluent reached 130.77 g/L ethanol while an average 8.18 g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.

  5. Use of a novel immobilization yeast system for winemaking.

    PubMed

    Peinado, Rafael A; Moreno, Juan J; Maestre, Oscar; Mauricio, Juan C

    2005-09-01

    Penicillium was used to immobilize Saccharomyces cerevisiae, without using physico-chemical external supports, to form yeast biocapsules. The biocapsules, once the Penicillium was killed by the ethanol produced, were used in a grape must fermentation. Must fermentation was carried out for 160 h with the biocapsules and for 300 h with free yeast cells. Acetaldehyde (84 vs. 63 mg/l), isobutanol (217 vs. 194 mg/l), L: -proline (7.7 vs. 6.5 mM: ) and aspartic acid (0.42 vs. 0 mM: ) in final wine were higher with the biocapsules than with free cells.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. Drug screening using model systems: some basics

    PubMed Central

    2016-01-01

    ABSTRACT An increasing number of laboratories that focus on model systems are considering drug screening. Executing a drug screen is complicated enough. But the path for moving initial hits towards the clinic requires a different knowledge base and even a different mindset. In this Editorial I discuss the importance of doing some homework before you start screening. 'Lead hits', 'patentable chemical space' and 'druggability' are all concepts worth exploring when deciding which screening path to take. I discuss some of the lessons I learned that may be useful as you navigate the screening matrix. PMID:27821602

  13. [The yeast biofilm in human medicine].

    PubMed

    Růzicka, Filip; Holá, Veronika; Votava, Miroslav

    2007-08-01

    In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastrointestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to

  14. 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...

  15. 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.

  16. Newborn screening healthcare information system based on service-oriented architecture.

    PubMed

    Hsieh, Sung-Huai; Hsieh, Sheau-Ling; Chien, Yin-Hsiu; Weng, Yung-Ching; Hsu, Kai-Ping; Chen, Chi-Huang; Tu, Chien-Ming; Wang, Zhenyu; Lai, Feipei

    2010-08-01

    In this paper, we established a newborn screening system under the HL7/Web Services frameworks. We rebuilt the NTUH Newborn Screening Laboratory's original standalone architecture, having various heterogeneous systems operating individually, and restructured it into a Service-Oriented Architecture (SOA), distributed platform for further integrity and enhancements of sample collections, testing, diagnoses, evaluations, treatments or follow-up services, screening database management, as well as collaboration, communication among hospitals; decision supports and improving screening accuracy over the Taiwan neonatal systems are also addressed. In addition, the new system not only integrates the newborn screening procedures among phlebotomy clinics, referral hospitals, as well as the newborn screening center in Taiwan, but also introduces new models of screening procedures for the associated, medical practitioners. Furthermore, it reduces the burden of manual operations, especially the reporting services, those were heavily dependent upon previously. The new system can accelerate the whole procedures effectively and efficiently. It improves the accuracy and the reliability of the screening by ensuring the quality control during the processing as well.

  17. 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

  18. 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

  19. Physiological and environmental control of yeast prions

    PubMed Central

    Chernova, Tatiana A.; Wilkinson, Keith D.; Chernoff, Yury O.

    2014-01-01

    Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions. PMID:24236638

  20. Functional Analysis With a Barcoder Yeast Gene Overexpression System

    PubMed Central

    Douglas, Alison C.; Smith, Andrew M.; Sharifpoor, Sara; Yan, Zhun; Durbic, Tanja; Heisler, Lawrence E.; Lee, Anna Y.; Ryan, Owen; Göttert, Hendrikje; Surendra, Anu; van Dyk, Dewald; Giaever, Guri; Boone, Charles; Nislow, Corey; Andrews, Brenda J.

    2012-01-01

    Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions. PMID:23050238

  1. The yin and yang of yeast: biodiversity research and systems biology as complementary forces driving innovation in biotechnology.

    PubMed

    Roberts, Ian N; Oliver, Stephen G

    2011-03-01

    The aim of this article is to review how yeast has contributed to contemporary biotechnology and to seek underlying principles relevant to its future exploitation for human benefit. Recent advances in systems biology combined with new knowledge of genome diversity promise to make yeast the eukaryotic workhorse of choice for production of everything from probiotics and pharmaceuticals to fuels and chemicals. The ability to engineer new capabilities through introduction of controlled diversity based on a complete understanding of genome complexity and metabolic flux is key. Here, we briefly summarise the history that has led to these apparently simple organisms being employed in such a broad range of commercial applications. Subsequently, we discuss the likely consequences of current yeast research for the future of biotechnological innovation.

  2. 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.

  3. Development of lung cancer CT screening operating support system

    NASA Astrophysics Data System (ADS)

    Ishigaki, Rikuta; Hanai, Kozou; Suzuki, Masahiro; Kawata, Yoshiki; Niki, Noboru; Eguchi, Kenji; Kakinuma, Ryutaro; Moriyama, Noriyuki

    2009-02-01

    In Japan, lung cancer death ranks first among men and third among women. Lung cancer death is increasing yearly, thus early detection and treatment are needed. For this reason, CT screening for lung cancer has been introduced. The CT screening services are roughly divided into three sections: office, radiology and diagnosis sections. These operations have been performed through paper-based or a combination of paper-based and an existing electronic health recording system. This paper describes an operating support system for lung cancer CT screening in order to make the screening services efficient. This operating support system is developed on the basis of 1) analysis of operating processes, 2) digitalization of operating information, and 3) visualization of operating information. The utilization of the system is evaluated through an actual application and users' survey questionnaire obtained from CT screening centers.

  4. [Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples].

    PubMed

    Gayibova, Ülkü; Dalyan Cılo, Burcu; Ağca, Harun; Ener, Beyza

    2014-07-01

    Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C

  5. Meeting Report on Experimental Approaches to Evolution and Ecology Using Yeast and Other Model Systems

    PubMed Central

    Jarosz, Daniel F.; Dudley, Aimée M.

    2017-01-01

    The fourth EMBO-sponsored conference on Experimental Approaches to Evolution and Ecology Using Yeast and Other Model Systems (https://www.embl.de/training/events/2016/EAE16-01/), was held at the EMBL in Heidelberg, Germany, October 19–23, 2016. The conference was organized by Judith Berman (Tel Aviv University), Maitreya Dunham (University of Washington), Jun-Yi Leu (Academia Sinica), and Lars Steinmetz (EMBL Heidelberg and Stanford University). The meeting attracted ∼120 researchers from 28 countries and covered a wide range of topics in the fields of genetics, evolutionary biology, and ecology, with a unifying focus on yeast as a model system. Attendees enjoyed the Keith Haring-inspired yeast florescence microscopy artwork (Figure 1), a unique feature of the meeting since its inception, and the 1 min flash talks that catalyzed discussions at two vibrant poster sessions. The meeting coincided with the 20th anniversary of the publication describing the sequence of the first eukaryotic genome, Saccharomyces cerevisiae. Many of the conference talks focused on important questions about what is contained in the genome, how genomes evolve, and the architecture and behavior of communities of phenotypically and genotypically diverse microorganisms. Here, we summarize highlights of the research talks around these themes. Nearly all presentations focused on novel findings, and we refer the reader to relevant manuscripts that have subsequently been published. PMID:28814445

  6. 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

  7. 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.

  8. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-02

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

  9. 14 CFR 23.1105 - Induction system screens.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... upstream of the carburetor or fuel injection system. (b) No screen may be in any part of the induction... alcohol alone; and (d) It must be impossible for fuel to strike any screen. [Doc. No. 4080, 29 FR 17955...

  10. 14 CFR 23.1105 - Induction system screens.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... upstream of the carburetor or fuel injection system. (b) No screen may be in any part of the induction... alcohol alone; and (d) It must be impossible for fuel to strike any screen. [Doc. No. 4080, 29 FR 17955...

  11. 14 CFR 23.1105 - Induction system screens.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... upstream of the carburetor or fuel injection system. (b) No screen may be in any part of the induction... alcohol alone; and (d) It must be impossible for fuel to strike any screen. [Doc. No. 4080, 29 FR 17955...

  12. 14 CFR 23.1105 - Induction system screens.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... upstream of the carburetor or fuel injection system. (b) No screen may be in any part of the induction... alcohol alone; and (d) It must be impossible for fuel to strike any screen. [Doc. No. 4080, 29 FR 17955...

  13. 14 CFR 23.1105 - Induction system screens.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... upstream of the carburetor or fuel injection system. (b) No screen may be in any part of the induction... alcohol alone; and (d) It must be impossible for fuel to strike any screen. [Doc. No. 4080, 29 FR 17955...

  14. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System

    PubMed Central

    Hoffman, Charles S.; Wood, Valerie; Fantes, Peter A.

    2015-01-01

    The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe. PMID:26447128

  15. Unravel lipid accumulation mechanism in oleaginous yeast through single cell systems biology study

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

    Xie, Xiaoliang; Ding, Shiyou

    Searching for alternative and clean energy is one of the most important tasks today. Our research aimed at finding the best living condition for certain types of oleaginous yeasts for efficient lipid production. We found that R. glutinis yeast cells has great variability in lipid production among cells while Y. lipolytica cells has similar oil production ability. We found some individual cells shows much higher level of oil production. In order to further study these cases, we employed a label-free chemical sensitive microscopy method call stimulated Raman scattering (SRS). With SRS, we could measure the lipid content in each cell.more » We combined SRS microscopy with microfluidic device so that we can isolate cells with high fat content. We also developed SRS imaging technique that has higher imaging speed, which is highly desirable for high throughput cell screening and sorting. Since these cells has similar genome, it must be the transcriptome caused their difference in oil production. We developed a single cell transcriptome sequencing method to study which genes are responsible for elevated oil production. These methods that are developed for this project can easily be applied for many other areas of research. For example, the single transcriptome can be used to study the transcriptomes of other cell types. The high-speed SRS microscopy techniques can be used to speed up chemical imaging for lablefree histology or imaging distribution of chemicals in tissues of live mice or in humans. The developed microfluidic platform can be used to sort other type of cells, e.g., white blood cells for diagnosis of cancer or other blood diseases.« less

  16. Evolutionary biology through the lens of budding yeast comparative genomics.

    PubMed

    Marsit, Souhir; Leducq, Jean-Baptiste; Durand, Éléonore; Marchant, Axelle; Filteau, Marie; Landry, Christian R

    2017-10-01

    The budding yeast Saccharomyces cerevisiae is a highly advanced model system for studying genetics, cell biology and systems biology. Over the past decade, the application of high-throughput sequencing technologies to this species has contributed to this yeast also becoming an important model for evolutionary genomics. Indeed, comparative genomic analyses of laboratory, wild and domesticated yeast populations are providing unprecedented detail about many of the processes that govern evolution, including long-term processes, such as reproductive isolation and speciation, and short-term processes, such as adaptation to natural and domestication-related environments.

  17. 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

  18. Nectar yeasts: a natural microcosm for ecology.

    PubMed

    Chappell, Callie R; Fukami, Tadashi

    2018-06-01

    The species of yeasts that colonize floral nectar can modify the mutualistic relationships between plants and pollinators by changing the chemical properties of nectar. Recent evidence supporting this possibility has led to increased interest among ecologists in studying these fungi as well as the bacteria that interact with them in nectar. Although not fully explored, nectar yeasts also constitute a promising natural microcosm that can be used to facilitate development of general ecological theory. We discuss the methodological and conceptual advantages of using nectar yeasts from this perspective, including simplicity of communities, tractability of dispersal, replicability of community assembly, and the ease with which the mechanisms of species interactions can be studied in complementary experiments conducted in the field and the laboratory. To illustrate the power of nectar yeasts as a study system, we discuss several topics in community ecology, including environmental filtering, priority effects, and metacommunity dynamics. An exciting new direction is to integrate metagenomics and comparative genomics into nectar yeast research to address these fundamental ecological topics. Copyright © 2018 John Wiley & Sons, Ltd.

  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. 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.

  2. 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

  3. 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.

  4. Yeast biotechnology: teaching the old dog new tricks.

    PubMed

    Mattanovich, Diethard; Sauer, Michael; Gasser, Brigitte

    2014-03-06

    Yeasts are regarded as the first microorganisms used by humans to process food and alcoholic beverages. The technology developed out of these ancient processes has been the basis for modern industrial biotechnology. Yeast biotechnology has gained great interest again in the last decades. Joining the potentials of genomics, metabolic engineering, systems and synthetic biology enables the production of numerous valuable products of primary and secondary metabolism, technical enzymes and biopharmaceutical proteins. An overview of emerging and established substrates and products of yeast biotechnology is provided and discussed in the light of the recent literature.

  5. 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

  6. 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.

  7. 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

  8. Tapping into yeast diversity.

    PubMed

    Fay, Justin C

    2012-11-01

    Domesticated organisms demonstrate our capacity to influence wild species but also provide us with the opportunity to understand rapid evolution in the context of substantially altered environments and novel selective pressures. Recent advances in genetics and genomics have brought unprecedented insights into the domestication of many organisms and have opened new avenues for further improvements to be made. Yet, our ability to engineer biological systems is not without limits; genetic manipulation is often quite difficult. The budding yeast, Saccharomyces cerevisiae, is not only one of the most powerful model organisms, but is also the premier producer of fermented foods and beverages around the globe. As a model system, it entertains a hefty workforce dedicated to deciphering its genome and the function it encodes at a rich mechanistic level. As a producer, it is used to make leavened bread, and dozens of different alcoholic beverages, such as beer and wine. Yet, applying the awesome power of yeast genetics to understanding its origins and evolution requires some knowledge of its wild ancestors and the environments from which they were derived. A number of surprisingly diverse lineages of S. cerevisiae from both primeval and secondary forests in China have been discovered by Wang and his colleagues. These lineages substantially expand our knowledge of wild yeast diversity and will be a boon to elucidating the ecology, evolution and domestication of this academic and industrial workhorse.

  9. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System.

    PubMed

    Hoffman, Charles S; Wood, Valerie; Fantes, Peter A

    2015-10-01

    The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe. Copyright © 2015 by the Genetics Society of America.

  10. 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

  11. 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

  12. 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.

  13. Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

    PubMed Central

    Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

    2012-01-01

    Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

  14. 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

  15. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  16. 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.

  17. Assessment of yeast as a dietary additive on haematology and water quality of common carp in a recirculating aquaculture system

    NASA Astrophysics Data System (ADS)

    Goran, Siraj Muhammed Abdulla; Omar, Samad Sofy; Anwer, Ayub Youns

    2017-09-01

    Feeding experiment was accomplished at the Aquaculture unit (Close system), Grdarasha station, Agriculture College, University of Salahaddin, Erbil, Kurdistan Region, to investigate different levels of Aquagrow E (AGEY) brewer's yeast cell Saccharomyces cerevisiae on the haematological and water quality of common carp fingerlings Cyprinus carpio. The basal diet was formulated to contain 34% protein and 10% lipid and the dietary treatments were supplemented with 0.5%, 1% and 1.5% of AGEY diet. A total of 180 Common carp (10.30 ± 0.27 g) fed on experimental diets for 10 weeks. Water quality assessment for well water and pond water for rearing Cyprinus carpio in cage system conducted weekly, while some parameters including pH, EC, water temperature and DO were monitored daily during the entire periods of study. Values of total hardness, alkalinity, ammonia and nitrate for studied water samples were within normal ranges for rearing Cyprinus carpio. Mean concentration of GPT, GOT and Glucose were 104 to 170 U/L, 1371 to 3308 U/L and 34 to 63mg/dl respectively, moreover, highest levels were observed in treatments with higher concentrations of yeast in its food except for blood sugar. Slight variation in lipase enzyme were found between control and treatment groups, while levels of amylase enzyme were increased toward cages with higher levels of yeast until T1 and then decreased toward T3. Total protein levels were increased to toward higher levels of yeast in food of Cyprinus carpio fish. Haematological results showed highest levels of WBC and platelets in treatments cages than control group. Levels of RBCs and hemoglobin were highest in treatment group 1 with 0.5%of yeast than treatments higher yeast concentrations. Significant correlation was found in haematological parameters between control and treatments.

  18. Heat Shock Partially Dissociates the Overlapping Modules of the Yeast Protein-Protein Interaction Network: A Systems Level Model of Adaptation

    PubMed Central

    Mihalik, Ágoston; Csermely, Peter

    2011-01-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a ‘stratus-cumulus’ type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

  19. Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory.

    PubMed

    Edwards, Thea M; Morgan, Howard E; Balasca, Coralia; Chalasani, Naveen K; Yam, Lauren; Roark, Alison M

    2018-01-01

    The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade "Environmental Estrogens" (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ERα) and/or beta (ERβ). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-β-D-galactopyranoside (ONPG) or chlorophenol red-β-D-galactopyranoside (CPRG)) that are cleaved by β-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting

  20. Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

    PubMed Central

    Edwards, Thea M.; Morgan, Howard E.; Balasca, Coralia; Chalasani, Naveen K.; Yam, Lauren; Roark, Alison M.

    2018-01-01

    The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade "Environmental Estrogens" (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ERα) and/or beta (ERβ). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-β-D-galactopyranoside (ONPG) or chlorophenol red-β-D-galactopyranoside (CPRG)) that are cleaved by β-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting

  1. Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

    PubMed

    Malcov, Mira; Cesarkas, Karen; Stelzer, Gil; Shalom, Sarah; Dicken, Yosef; Naor, Yaniv; Goldstein, Ronald S; Sagee, Shira; Kassir, Yona; Don, Jeremy

    2004-12-01

    Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

  2. 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.

  3. Yeast biotechnology: teaching the old dog new tricks

    PubMed Central

    2014-01-01

    Yeasts are regarded as the first microorganisms used by humans to process food and alcoholic beverages. The technology developed out of these ancient processes has been the basis for modern industrial biotechnology. Yeast biotechnology has gained great interest again in the last decades. Joining the potentials of genomics, metabolic engineering, systems and synthetic biology enables the production of numerous valuable products of primary and secondary metabolism, technical enzymes and biopharmaceutical proteins. An overview of emerging and established substrates and products of yeast biotechnology is provided and discussed in the light of the recent literature. PMID:24602262

  4. Functional purification of the monocarboxylate transporter of the yeast Candida utilis.

    PubMed

    Baltazar, Fátima; Cássio, Fernanda; Leão, Cecília

    2006-08-01

    Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-beta-D-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.

  5. L-arabinose fermenting yeast

    DOEpatents

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

    2010-12-07

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

  6. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

    PubMed

    Tipper, Donald J; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  7. Anti-Saccharomyces cerevisiae antibodies (ASCA) are associated with body fat mass and systemic inflammation, but not with dietary yeast consumption: a cross-sectional study.

    PubMed

    Kvehaugen, Anne Stine; Aasbrenn, Martin; Farup, Per G

    2017-01-01

    Baker's/brewer's yeast, Saccharomyces cerevisiae , has been used as an alternative to antibiotic growth promoters to improve growth performance in animals. In humans, Saccharomyces cerevisiae is among the most commonly detected fungi in fecal samples and likely originates from food. Recently, an association between anti- Saccharomyces cerevisiae antibodies (ASCA) and obesity in humans was suggested, but the cause of the elevated ASCA levels is not clear. Our aim was to study ASCA in morbidly obese subjects and explore potential associations with anthropometrics, diet, co-morbidities and biomarkers of inflammation and gut permeability. Subjects with morbid obesity referred to a specialized hospital unit were included. Diet and clinical data were recorded with self-administered questionnaires. Main dietary sources of baker's/brewer's yeast (e.g. bread and beer) were used as a proxy for the intake of yeast. Laboratory analyses included ASCA, serum zonulin (reflecting gut permeability), C-reactive protein and a routine haematological and biochemical screening. One-hundred-and-forty subjects; 109 (78%) female, 98 with dietary records, mean age 43 years and BMI 42 kg/m 2 were included. The number of ASCA positive subjects was 31 (22%) for IgG, 4 (2.9%) for IgA and 3 (2.1%) for IgM. Age, body fat mass and C-reactive protein were significantly higher in IgG-positive compared to IgG-negative subjects ( P  < 0.05). A borderline significant association was found between elevated zonulin and ASCA IgG-positivity ( P  = 0.06). No association was found between yeast-containing food and ASCA IgG-positivity, or between yeast-containing food and fat mass. The findings indicate that ASCA IgG-positivity may be linked to the generalized inflammation commonly seen with increased adiposity, but not to dietary yeast intake. Other potential causes for the raised ASCA IgG concentrations, such as genetic predisposition, deviations in the gut microbiota and cross-reactivity of ASCA with

  8. Genome dynamics and evolution in yeasts: A long-term yeast-bacteria competition experiment

    PubMed Central

    Katz, Michael; Knecht, Wolfgang; Compagno, Concetta; Piškur, Jure

    2018-01-01

    There is an enormous genetic diversity evident in modern yeasts, but our understanding of the ecological basis of such diversifications in nature remains at best fragmented so far. Here we report a long-term experiment mimicking a primordial competitive environment, in which yeast and bacteria co-exist and compete against each other. Eighteen yeasts covering a wide phylogenetic background spanning approximately 250 million years of evolutionary history were used to establish independent evolution lines for at most 130 passages. Our collection of hundreds of modified strains generated through such a rare two-species cross-kingdom competition experiment re-created the appearance of large-scale genomic rearrangements and altered phenotypes important in the diversification history of yeasts. At the same time, the methodology employed in this evolutionary study would also be a non-gene-technological method of reprogramming yeast genomes and then selecting yeast strains with desired traits. Cross-kingdom competition may therefore be a method of significant value to generate industrially useful yeast strains with new metabolic traits. PMID:29624585

  9. The ecology of the Drosophila-yeast mutualism in wineries

    PubMed Central

    2018-01-01

    The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila. PMID:29768432

  10. The ecology of the Drosophila-yeast mutualism in wineries.

    PubMed

    Quan, Allison S; Eisen, Michael B

    2018-01-01

    The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila.

  11. Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve Malonyl-CoA-Derived Product.

    PubMed

    Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2018-01-01

    Malonyl-coenzyme A (malonyl-CoA) is a critical precursor for the biosynthesis of a variety of biochemicals. It is synthesized by the catalysis of acetyl-CoA carboxylase (Acc1p), which was demonstrated to be deactivated by the phosphorylation of Snf1 protein kinase in yeast. In this study, we designed a synthetic malonyl-CoA biosensor and used it to screen phosphorylation site mutations of Acc1p in Saccharomyces cerevisiae . Thirteen phosphorylation sites were mutated, and a combination of three site mutations in Acc1p, S686A, S659A, and S1157A, was found to increase malonyl-CoA availability. ACC1 S686AS659AS1157A expression also improved the production of 3-hydroxypropionic acid, a malonyl-CoA-derived chemical, compared to both wild type and the previously reported ACC1 S659AS1157A mutation. This mutation will also be beneficial for other malonyl-CoA-derived products.

  12. System Strategies for Colorectal Cancer Screening at Federally Qualified Health Centers

    PubMed Central

    Levy, Barcey T.; Moss, Carol A.; Bay, Camden P.

    2015-01-01

    Objectives. We assessed the protocols and system processes for colorectal cancer (CRC) screening at federally qualified health centers (FQHCs) in 4 midwestern states. Methods. We identified 49 FQHCs in 4 states. In January 2013, we mailed their medical directors a 49-item questionnaire about policies on CRC screening, use of electronic medical records, types of CRC screening recommended, clinic tracking systems, referrals for colonoscopy, and barriers to providing CRC. Results. Forty-four questionnaires (90%) were returned. Thirty-three of the respondents (75%) estimated the proportion of their patients up-to-date with CRC screening, with a mean of 35%. One major barrier to screening was inability to provide colonoscopy for patients with a positive fecal occult blood test (59%). The correlation of system strategies and estimated percentage of patients up-to-date with CRC screening was 0.43 (P = .01). Conclusions. CRC system strategies were associated with higher CRC screening rates. Implementing system strategies for CRC screening takes time and effort and is important to maintain, to help prevent, or to cure many cases of CRC, the second leading cause of cancer in the United States. PMID:24832146

  13. Screening Saccharomyces cerevisiae Distillery Strains in Industrial Media

    USDA-ARS?s Scientific Manuscript database

    Twenty-four distillery yeast strains were obtained from the ARS Culture Collection (NRRL) in Peoria, IL, and screened for ethanol production at 30 and 35°C using industrial media. The medium used in the tests consisted of corn mash prepared by combining coarse ground corn, water, and stillage from a...

  14. [A comparative study between the Vitek YBC and Microscan Walk Away RYID automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest].

    PubMed

    Ferrara, Giuseppe; Mercedes Panizol, Maria; Mazzone, Marja; Delia Pequeneze, Maria; Reviakina, Vera

    2014-12-01

    The aim of this study was to compare the identification of clin- ically relevant yeasts by the Vitek YBC and Microscan Walk Away RYID automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2 x 2 contingency tables, McNemar's Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: (1) frequent isolation and (2) rare isolation. The Vitek YBC and Microscan Walk Away RYID systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.

  15. 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.

  16. Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals

    PubMed Central

    Shi, Shuobo; Zhao, Huimin

    2017-01-01

    Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium, Trichosporon, and Lipomyces. This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years. PMID:29167664

  17. Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals.

    PubMed

    Shi, Shuobo; Zhao, Huimin

    2017-01-01

    Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium , Trichosporon , and Lipomyces . This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years.

  18. Association between Grape Yeast Communities and the Vineyard Ecosystems

    PubMed Central

    Drumonde-Neves, João; Lima, Teresa; Schuller, Dorit; Pais, Célia

    2017-01-01

    The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Açores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viticultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions. PMID:28085916

  19. Responses of Yeast Biocontrol Agents to Environmental Stress

    PubMed Central

    Sui, Yuan; Wisniewski, Michael; Droby, Samir

    2015-01-01

    Biological control of postharvest diseases, utilizing wild species and strains of antagonistic yeast species, is a research topic that has received considerable attention in the literature over the past 30 years. In principle, it represents a promising alternative to chemical fungicides for the management of postharvest decay of fruits, vegetables, and grains. A yeast-based biocontrol system is composed of a tritrophic interaction between a host (commodity), a pathogen, and a yeast species, all of which are affected by environmental factors such as temperature, pH, and UV light as well as osmotic and oxidative stresses. Additionally, during the production process, biocontrol agents encounter various severe abiotic stresses that also impact their viability. Therefore, understanding the ecological fitness of the potential yeast biocontrol agents and developing strategies to enhance their stress tolerance are essential to their efficacy and commercial application. The current review provides an overview of the responses of antagonistic yeast species to various environmental stresses, the methods that can be used to improve stress tolerance and efficacy, and the related mechanisms associated with improved stress tolerance. PMID:25710368

  20. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

    PubMed

    Yeung, Yik A; Wittrup, K Dane

    2002-01-01

    Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

  1. A proposal for cervical screening information systems in developing countries.

    PubMed

    Marrett, Loraine D; Robles, Sylvia; Ashbury, Fredrick D; Green, Bo; Goel, Vivek; Luciani, Silvana

    2002-11-20

    The effective and efficient delivery of cervical screening programs requires information for planning, management, delivery and evaluation. Specially designed systems are generally required to meet these needs. In many developing countries, lack of information systems constitutes an important barrier to development of comprehensive screening programs and the effective control of cervical cancer. Our report outlines a framework for creating such systems in developing countries and describes a conceptual model for a cervical screening information system. The proposed system is modular, recognizing that there will be considerable between-region heterogeneity in current status and priorities. The proposed system is centered on modules that would allow for the assembly and computerization of data on Pap tests, since these represent the main screening modality at the present time. Additional modules would process data and create and maintain a screening database (e.g., standardize, edit, link and update modules) and allow for the integration of other types of data, such as cervical histopathology results. An open systems development model is proposed, since it is most compatible with the goals of local stakeholder involvement and capacity-building. Copyright 2002 Wiley-Liss, Inc.

  2. Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Scheffersomyces stipitis is a Crabtree negative yeast, commonly known for its capacity to ferment pentose sugars. Differently from Crabtree positive yeasts such as Saccharomyces cerevisiae, the onset of fermentation in S. stipitis is not dependent on the sugar concentration, but is regulated by a decrease in oxygen levels. Even though S. stipitis has been extensively studied due to its potential application in pentoses fermentation, a limited amount of information is available about its metabolism during aerobic growth on glucose. Here, we provide a systems biology based comparison between the two yeasts, uncovering the metabolism of S. stipitis during aerobic growth on glucose under batch and chemostat cultivations. Results Starting from the analysis of physiological data, we confirmed through 13C-based flux analysis the fully respiratory metabolism of S. stipitis when growing both under glucose limited or glucose excess conditions. The patterns observed showed similarity to the fully respiratory metabolism observed for S. cerevisiae under chemostat cultivations however, intracellular metabolome analysis uncovered the presence of several differences in metabolite patterns. To describe gene expression levels under the two conditions, we performed RNA sequencing and the results were used to quantify transcript abundances of genes from the central carbon metabolism and compared with those obtained with S. cerevisiae. Interestingly, genes involved in central pathways showed different patterns of expression, suggesting different regulatory networks between the two yeasts. Efforts were focused on identifying shared and unique families of transcription factors between the two yeasts through in silico transcription factors analysis, suggesting a different regulation of glycolytic and glucoenogenic pathways. Conclusions The work presented addresses the impact of high-throughput methods in describing and comparing the physiology of Crabtree positive and Crabtree

  3. The yeasts phosphorelay systems: a comparative view.

    PubMed

    Salas-Delgado, Griselda; Ongay-Larios, Laura; Kawasaki-Watanabe, Laura; López-Villaseñor, Imelda; Coria, Roberto

    2017-06-01

    Cells contain signal transduction pathways that mediate communication between the extracellular environment and the cell interior. These pathways control transcriptional programs and posttranscriptional processes that modify cell metabolism in order to maintain homeostasis. One type of these signal transduction systems are the so-called Two Component Systems (TCS), which conduct the transfer of phosphate groups between specific and conserved histidine and aspartate residues present in at least two proteins; the first protein is a sensor kinase which autophosphorylates a histidine residue in response to a stimulus, this phosphate is then transferred to an aspartic residue located in a response regulator protein. There are classical and hybrid TCS, whose difference consists in the number of proteins and functional domains involved in the phosphorelay. The TCS are widespread in bacteria where the sensor and its response regulator are mostly specific for a given stimulus. In eukaryotic organisms such as fungi, slime molds, and plants, TCS are present as hybrid multistep phosphorelays, with a variety of arrangements (Stock et al. in Annu Rev Biochem 69:183-215, 2000; Wuichet et al. in Curr Opin Microbiol 292:1039-1050, 2010). In these multistep phosphorelay systems, several phosphotransfer events take place between different histidine and aspartate residues localized in specific domains present in more than two proteins (Thomason and Kay, in J Cell Sci 113:3141-3150, 2000; Robinson et al. in Nat Struct Biol 7:626-633, 2000). This review presents a brief and succinct description of the Two-component systems of model yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans and Kluyveromyces lactis. We have focused on the comparison of domain organization and functions of each component present in these phosphorelay systems.

  4. Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal.

    PubMed

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

    2012-04-01

    Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from "Mezcal de Oaxaca" were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave tequilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. 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

  6. 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.

  7. The impact of yeast fermentation on dough matrix properties.

    PubMed

    Rezaei, Mohammad N; Jayaram, Vinay B; Verstrepen, Kevin J; Courtin, Christophe M

    2016-08-01

    Most studies on dough properties are performed on yeastless dough to exclude the complicating, time-dependent effect of yeast. Baker's yeast, however, impacts dough matrix properties during fermentation, probably through the production of primary (CO2 and ethanol) and secondary (glycerol, acetic acid and succinic acid) metabolites. The aim of this study is to obtain a better understanding of the changes in yeasted dough behavior introduced by fermentation, by investigating the impact of yeast fermentation on Farinograph dough consistency, dough spread, Kieffer rig dough extensibility and gluten agglomeration behavior in a fermented dough-batter gluten starch separation system. Results show that fermentation leads to a dough with less flow and lower extensibility that breaks more easily under stress and strain. The dough showed less elastic and more plastic deformation behavior. Gluten agglomerates were smaller for yeasted dough than for the unyeasted control. These changes probably have to be attributed to metabolites generated during fermentation. Indeed, organic acids and also ethanol in concentrations produced by yeast were previously shown to have similar effects in yeastless dough. These findings imply the high importance of yeast fermentation metabolites on dough matrix properties in industrial bread production. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

    PubMed

    Patel, Chirag A; Wang, Jinqing; Wang, Xinwei; Dong, Feng; Zhong, Pingyu; Luo, Peter P; Wang, Kevin C

    2011-09-01

    We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

  9. Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

    PubMed Central

    Noguchi, Chiemi; Watanabe, Daisuke; Zhou, Yan; Akao, Takeshi

    2012-01-01

    Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains. PMID:22057870

  10. Association of constitutive hyperphosphorylation of Hsf1p with a defective ethanol stress response in Saccharomyces cerevisiae sake yeast strains.

    PubMed

    Noguchi, Chiemi; Watanabe, Daisuke; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

    2012-01-01

    Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

  11. Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway

    PubMed Central

    1994-01-01

    Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy. PMID:8051211

  12. Klebsiella pneumoniae type 3 fimbriae agglutinate yeast in a mannose-resistant manner.

    PubMed

    Stahlhut, Steen G; Struve, Carsten; Krogfelt, Karen A

    2012-03-01

    The ability of bacterial pathogens to express different fimbrial adhesins plays a significant role in virulence. Thus, specific detection of fimbrial expression is an important task in virulence characterization and epidemiological studies. Most clinical Klebsiella pneumoniae isolates express type 1 and type 3 fimbriae, which are characterized by mediation of mannose-sensitive agglutination of yeast cells and agglutination of tannic acid-treated ox red blood cells (RBCs), respectively. It has been observed that K. pneumoniae isolates agglutinate yeast cells and commercially available sheep RBCs in a mannose-resistant manner. Thus, this study was initiated to identify the adhesin involved. Screening of a mutant library surprisingly revealed that the mannose-resistant agglutination of yeast and sheep RBCs was mediated by type 3 fimbriae. Specific detection of type 1 fimbriae expression in K. pneumoniae was feasible only by the use of guinea pig RBCs. This was further verified by the use of isogenic fimbriae mutants and by cloning and expressing K. pneumoniae fimbrial gene clusters in Escherichia coli. Yeast agglutination assays are commonly used to detect type 1 fimbriae expression but should not be used for bacterial species able to express type 3 fimbriae. For these species, the use of guinea pig blood for specific type 1 fimbriae detection is essential. The use of commercially available sheep RBCs or yeast is an easy alternative to traditional methods to detect type 3 fimbriae expression. Easy and specific detection of expression of type 1 and type 3 fimbriae is essential in the continuous characterization of these important adhesive virulence factors present in members of the Enterobacteriaceae.

  13. Vaginal yeast infection

    MedlinePlus

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

  14. Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4+ counts

    PubMed Central

    Kumar, Deepa Anil; Muralidhar, Sumathi; Banerjee, Uma; Basir, Seemi Farhat; Mathur, Purva; Khan, Luqman Ahmad

    2015-01-01

    Background: Yeasts are important opportunistic pathogens, in individuals infected with human immunodeficiency virus (HIV). Yeast species inhabiting the oral mucosa of HIV-infected persons can act as source of oral lesions, especially as the individual progresses towards immunocompromised state. Present study was conducted to evaluate the diversity of yeasts in oral cavities of asymptomatic HIV-infected persons and their association with CD4+ cell counts. Materials and Methods: 100 HIV seropositive subjects and 100 healthy controls were screened for oral yeast carriage using standard procedures. Results: Of the 100 HIV-seropositive persons screened, 48 were colonized by different yeasts, either alone or in association with another species. Candida albicans was the most common species (56.90%) while non C. albicans Candida (NCAC) accounted for 39.65%. Among NCAC, Candida tropicalis and Candida krusei were most common. One isolate each of rare opportunistic pathogenic yeasts, Geotrichum candidum and Saccharomyces cereviseae, was recovered. The control group had an oral candidal carriage rate of 23%; C. albicans was the predominant species, followed by Candida glabrata, C. tropicalis and Candida parapsilosis. Antifungal susceptibility testing revealed no resistance in C. albicans, to the commonly used antifungal agents, whereas resistance or dose dependent susceptibility to fluconazole was observed in some of the NCAC species. Conclusion: Oral carriage of opportunistic pathogenic yeasts was greater in HIV-seropositive persons heading towards immunocompromised state, as evidenced by their CD4+ cell count. The predominant yeast isolated in this study (C. albicans), was found to be susceptible to commonly used antifungals. PMID:26392655

  15. Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

    PubMed

    Niki, Hironori

    2014-03-01

    The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.

  16. Dielectric modelling of cell division for budding and fission yeast

    NASA Astrophysics Data System (ADS)

    Asami, Koji; Sekine, Katsuhisa

    2007-02-01

    The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast.

  17. Gravitational radiation from compact binary systems in screened modified gravity

    NASA Astrophysics Data System (ADS)

    Zhang, Xing; Liu, Tan; Zhao, Wen

    2017-05-01

    Screened modified gravity (SMG) is a kind of scalar-tensor theory with screening mechanisms, which can suppress the fifth force in dense regions and allow theories to evade the solar system and laboratory tests. In this paper, we investigate how the screening mechanisms in SMG affect the gravitational radiation damping effects, calculate in detail the rate of the energy loss due to the emission of tensor and scalar gravitational radiations, and derive their contributions to the change in the orbital period of the binary system. We find that the scalar radiation depends on the screened parameters and the propagation speed of scalar waves, and the scalar dipole radiation dominates the orbital decay of the binary system. For strongly self-gravitating bodies, all effects of scalar sector are strongly suppressed by the screening mechanisms in SMG. By comparing our results to observations of binary system PSR J 1738 +0333 , we place the stringent constraints on the screening mechanisms in SMG. As an application of these results, we focus on three specific models of SMG (chameleon, symmetron, and dilaton), and derive the constraints on the model parameters, respectively.

  18. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  19. The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

    PubMed Central

    Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

    2012-01-01

    To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

  20. Live Yeast and Yeast Cell Wall Supplements Enhance Immune Function and Performance in Food-Producing Livestock: A Review †,‡

    PubMed Central

    Broadway, Paul R.; Carroll, Jeffery A.; Burdick Sanchez, Nicole C.

    2015-01-01

    More livestock producers are seeking natural alternatives to antibiotics and antimicrobials, and searching for supplements to enhance growth performance, and general animal health and well-being. Some of the compounds currently being utilized and studied are live yeast and yeast-based products derived from the strain Saccharomyces cerevisiae. These products have been reported to have positive effects both directly and indirectly on the immune system and its subsequent biomarkers, thereby mitigating negative effects associated with stress and disease. These yeast-based products have also been reported to simultaneously enhance growth and performance by enhancing dry matter intake (DMI) and average daily gain (ADG) perhaps through the establishment of a healthy gastrointestinal tract. These products may be especially useful in times of potential stress such as during birth, weaning, early lactation, and during the receiving period at the feedlot. Overall, yeast supplements appear to possess the ability to improve animal health and metabolism while decreasing morbidity, thereby enhancing profitability of these animals. PMID:27682097

  1. 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.

  2. Marine yeast isolation and industrial application.

    PubMed

    Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

    2014-09-01

    Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. © 2014 The Authors FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  3. ScreenCube: A 3D Printed System for Rapid and Cost-Effective Chemical Screening in Adult Zebrafish.

    PubMed

    Monstad-Rios, Adrian T; Watson, Claire J; Kwon, Ronald Y

    2018-02-01

    Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. In this study, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using prefilled screening plates, the system enables dosing of 96 fish in ∼3 min, with a 10-fold reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in postembryonic models of development, disease, and regeneration.

  4. Effects of distillation system and yeast strain on the aroma profile of Albariño (Vitis vinifera L.) grape pomace spirits.

    PubMed

    Arrieta-Garay, Y; Blanco, P; López-Vázquez, C; Rodríguez-Bencomo, J J; Pérez-Correa, J R; López, F; Orriols, I

    2014-10-29

    Orujo is a traditional alcoholic beverage produced in Galicia (northwest Spain) from distillation of grape pomace, a byproduct of the winemaking industry. In this study, the effect of the distillation system (copper charentais alembic versus packed column) and the yeast strain (native yeast L1 versus commercial yeast L2) on the chemical and sensory characteristics of orujo obtained from Albariño (Vitis vinifera L.) grape pomace has been analyzed. Principal component analysis, with two components explaining 74% of the variance, is able to clearly differentiate the distillates according to distillation system and yeast strain. Principal component 1, mainly defined by C6-C12 esters, isoamyl octanoate, and methanol, differentiates L1 from L2 distillates. In turn, principal component 2, mainly defined by linear alcohols, linalool, and 1-hexenol, differentiates alembic from packed column distillates. In addition, an aroma descriptive test reveals that the distillate obtained with a packed column from a pomace fermented with L1 presented the highest positive general impression, which is associated with the highest fruity and smallest solvent aroma scores. Moreover, chemical analysis shows that use of a packed column increases average ethanol recovery by 12%, increases the concentration of C6-C12 esters by 25%, and reduces the concentration of higher alcohols by 21%. In turn, L2 yeast obtained lower scores in the alembic distillates aroma profile. In addition, with L1, 9% higher ethanol yields were achieved, and L2 distillates contained 34%-40% more methanol than L1 distillates.

  5. Determination of Glucose Concentration in Yeast Culture Medium

    NASA Astrophysics Data System (ADS)

    Hara, Seiichi; Kishimoto, Tomokazu; Muraji, Masafumi; Tsujimoto, Hiroaki; Azuma, Masayuki; Ooshima, Hiroshi

    The present paper describes a sensor for measuring the glucose concentration of yeast culture medium. The sensor determines glucose concentration by measuring the yield of hydrogen peroxide produced by glucose oxidase, which is monitored as luminescence using photomultiplier. The present sensor is able to measure low glucose concentration in media in which yeast cells keep respiration state. We herein describe the system and the characteristics of the glucose sensor.

  6. 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

  7. Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

    PubMed Central

    Sitepu, Irnayuli R.; Jin, Mingjie; Fernandez, J. Enrique; da Costa Sousa, Leonardo; Balan, Venkatesh; Boundy-Mills, Kyria L.

    2015-01-01

    Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40% of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Pre-culturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals. PMID:25052467

  8. Yeast ecology of Kombucha fermentation.

    PubMed

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

  9. 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

  10. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast Pichia anomala for aflatoxin reduction

    USDA-ARS?s Scientific Manuscript database

    Pichia anomala WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of mycotoxin in the food chain...

  11. Marine yeast isolation and industrial application

    PubMed Central

    Zaky, Abdelrahman Saleh; Tucker, Gregory A; Daw, Zakaria Yehia; Du, Chenyu

    2014-01-01

    Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields. PMID:24738708

  12. Smart Screening System (S3) In Taconite Processing

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

    Daryoush Allaei; Angus Morison; David Tarnowski

    2005-09-01

    key components include smart motor and associated electronics, resonators, and supporting structural elements. It is shown that the smart motors have an acceptable life and performance. Resonator (or motion amplifier) designs are selected based on the final system requirement and vibration characteristics. All the components for a fully functional prototype are fabricated. The development program is on schedule. The last semi-annual report described the process of FE model validation and correlation with experimental data in terms of dynamic performance and predicted stresses. It also detailed efforts into making the supporting structure less important to system performance. Finally, an introduction into the dry application concept was presented. Since then, the design refinement phase was completed. This has resulted in a Smart Screen design that meets performance targets both in the dry condition and with taconite slurry flow using PZT motors. Furthermore, this system was successfully demonstrated for the DOE and partner companies at the Coleraine Mineral Research Laboratory in Coleraine, Minnesota.« less

  13. Yeast prions are useful for studying protein chaperones and protein quality control.

    PubMed

    Masison, Daniel C; Reidy, Michael

    2015-01-01

    Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.

  14. Intensified fractionation of brewery yeast waste for the recovery of invertase using aqueous two-phase systems.

    PubMed

    De León-González, Grecia; González-Valdez, José; Mayolo-Deloisa, Karla; Rito-Palomares, Marco

    2016-11-01

    The potential recovery of high-value products from brewery yeast waste confers value to this industrial residue. Aqueous two-phase systems (ATPS) have demonstrated to be an attractive alternative for the primary recovery of biological products and are therefore suitable for the recovery of invertase from this residue. Sixteen different polyethylene glycol (PEG)-potassium phosphate ATPS were tested to evaluate the effects of PEG molecular weight (MW) and tie-line length (TLL) upon the partition behavior of invertase. Concentrations of crude extract from brewery yeast waste were then varied in the systems that presented the best behaviors to intensify the potential recovery of the enzyme. Results show that the use of a PEG MW 400 g mol -1 system with a TLL of 45.0% (w/w) resulted in an invertase bottom phase recovery with a purification factor of 29.5 and a recovery yield of up to 66.2% after scaling the system to a total weight of 15.0 g. This represents 15.1 mg of invertase per mL of processed bottom phase. With these results, a single-stage ATPS process for the recovery of invertase is proposed. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  15. Identification of Candidate Genes Involved in the Salt Tolerance of Date Palm (Phoenix dactylifera L.) Based on a Yeast Functional Bioassay.

    PubMed

    Patankar, Himanshu V; Al-Harrasi, Ibtisam; Al-Yahyai, Rashid; Yaish, Mahmoud W

    2018-06-01

    Although date palm is a relatively salt-tolerant plant, the molecular basis of this tolerance is complex and poorly understood. Therefore, this study aimed to identify the genes involved in salinity tolerance using a basic yeast functional bioassay. To achieve this, a date palm cDNA library was overexpressed in Saccharomyces cerevisiae cells. The expression levels of selected genes that make yeast cells tolerant to salt were subsequently validated in the leaf and root tissues of date palm seedlings using a quantitative PCR method. About 6000 yeast transformant cells were replica printed and screened on a synthetic minimal medium containing 1.0 M of NaCl. The screening results showed the presence of 62 salt-tolerant transformant colonies. Sequence analysis of the recombinant yeast plasmids revealed the presence of a group of genes with potential salt-tolerance functions, such as aquaporins (PIP), serine/threonine protein kinases (STKs), ethylene-responsive transcription factor 1 (ERF1), and peroxidases (PRX). The expression pattern of the selected genes endorsed the hypothesis that these genes may be involved in salinity tolerance, as they showed a significant (p < 0.05) overexpression trend in both the leaf and root tissues in response to salinity. The genes identified in this project are suitable candidates for the further functional characterization of date palms.

  16. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    PubMed

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  17. Metabolic engineering of yeast for lignocellulosic biofuel production.

    PubMed

    Jin, Yong-Su; Cate, Jamie Hd

    2017-12-01

    Production of biofuels from lignocellulosic biomass remains an unsolved challenge in industrial biotechnology. Efforts to use yeast for conversion face the question of which host organism to use, counterbalancing the ease of genetic manipulation with the promise of robust industrial phenotypes. Saccharomyces cerevisiae remains the premier host for metabolic engineering of biofuel pathways, due to its many genetic, systems and synthetic biology tools. Numerous engineering strategies for expanding substrate ranges and diversifying products of S. cerevisiae have been developed. Other yeasts generally lack these tools, yet harbor superior phenotypes that could be exploited in the harsh processes required for lignocellulosic biofuel production. These include thermotolerance, resistance to toxic compounds generated during plant biomass deconstruction, and wider carbon consumption capabilities. Although promising, these yeasts have yet to be widely exploited. By contrast, oleaginous yeasts such as Yarrowia lipolytica capable of producing high titers of lipids are rapidly advancing in terms of the tools available for their metabolic manipulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Wine yeasts for the future.

    PubMed

    Fleet, Graham H

    2008-11-01

    International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.

  19. [High-contrast resolution of film-screen systems in oral and maxillofacial radiology].

    PubMed

    Kaeppler, G; Reinert, S

    2007-11-01

    The aim was to determine differences in high-contrast resolution of film-screen systems used in dental panoramic and cephalometric radiography by calculating the modulation transfer function (MTF). The radiographs used to determine the MTF should be taken by the same x-ray units as those used for patient radiographs. The MTF was determined using a lead grid and according to DIN 6867-2 for 11 film-screen systems (speed 250, speed class 200 and 400) used in dental radiographic diagnostics. The optical density was measured using a microdensitometer developed by PTB. With 10% of the modulation transfer factor, newly developed film-screen systems (speed class 200 and 400) demonstrated a resolution of 4.9 to 6 line pairs per mm (panoramic radiography). In cephalometric radiography a film-screen system (speed class 400 and green-sensitive film) had a resolution of 4.2 line pairs per mm and surpassed two film-screen systems (speed class 400, resolution of 3 line pairs per mm, blue-sensitive films). The relevance of this study is underlined by the diagnostic reference doses defined in the German X-ray Ordinance (RöV) which are also intended for dentistry. Film-screen systems (speed 250, speed class 200) previously used in dental panoramic and cephalometric radiography can be replaced by newly developed film-screen systems (speed class 400). In dental radiography dose reductions are possible with film-screen systems (speed class 400) without impairing diagnostic accuracy. The introduction of newly developed film-screen systems (speed class 400) requires lower milliampere-seconds and therefore an adjustment of the x-ray units to lower milliampere settings.

  20. Discussion of teleomorphic and anamorphic Ascomycetous yeasts and yeast-like taxa

    USDA-ARS?s Scientific Manuscript database

    The relationship of ascomycetous yeasts with other members of the ascomycete fungi (Ascomycota) has been controversial for over 100 years. Because yeasts are morphologically simple, it was proposed that they represent primitive forms of ascomycetes (e.g., Guilliermond 1912). Alternatively, the ide...

  1. Growth control of the eukaryote cell: a systems biology study in yeast.

    PubMed

    Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David Cj; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom Pj; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

    2007-01-01

    Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the

  2. Growth control of the eukaryote cell: a systems biology study in yeast

    PubMed Central

    Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David CJ; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom PJ; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

    2007-01-01

    Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome

  3. Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts.

    PubMed

    Guo, Yi-Cheng; Zhang, Lin; Dai, Shao-Xing; Li, Wen-Xing; Zheng, Jun-Juan; Li, Gong-Hua; Huang, Jing-Fei

    2016-01-01

    Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage.

  4. Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts

    PubMed Central

    Dai, Shao-Xing; Li, Wen-Xing; Zheng, Jun-Juan; Li, Gong-Hua; Huang, Jing-Fei

    2016-01-01

    Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage. PMID:27152421

  5. 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.

  6. 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

  7. 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.

  8. Why screening rates vary between Korea and Japan--differences between two national healthcare systems.

    PubMed

    Goto, Rei; Hamashima, Chisato; Mun, Sunghyun; Lee, Won-Chul

    2015-01-01

    Both Japan and Korea provide population-based screening programs. However, screening rates are much higher in Korea than in Japan. To clarify the possible factors explaining the differences between these two countries, we analyzed the current status of the cancer screening and background healthcare systems. Population- based cancer screening in Korea is coordinated well with social health insurance under a unified insurer system. In Japan, there are over 3,000 insurers and coordinating a comprehensive strategy for cancer screening promotion has been very difficult. The public healthcare system also has influence over cancer screening. In Korea, public healthcare does not cover a wide range of services. Almost free cancer screening and subsidization for medical cost for cancers detected in population-screening provides high incentive to participation. In Japan, on the other hand, a larger coverage of medical services, low co-payment, and a lenient medical audit enables people to have cancer screening under public health insurance as well as the broad range of cancer screening. The implementation of evidence-based cancer screening programs may be largely dependent on the background healthcare system. It is important to understand the impacts of each healthcare system as a whole and to match the characteristics of a particular health system when designing an efficient cancer screening system.

  9. Forces in yeast flocculation

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  10. The yeast actin cytoskeleton.

    PubMed

    Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

    2014-03-01

    The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. 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

  12. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis...

  13. Evaluation of baker's yeast in honey using a real-time PCR assay.

    PubMed

    Kast, Christina; Roetschi, Alexandra

    2017-04-01

    Occasionally, melissopalynological analysis reveals the presence of baker's yeast (Saccharomyces cerevisiae) in honey sediments. A field experiment reproducing a common spring bee feeding practice, using sugar paste containing baker's yeast, was performed to understand how S. cerevisiae are introduced into honey. Apart from classical microscopy, a real-time quantitative PCR (qPCR) system specific for S. cerevisiae was established for quantification of S. cerevisiae in honeys. Results showed that S. cerevisiae cells are stored in the honey of the brood combs and are also transferred into honey in the supers. The concentrations of S. cerevisiae were highest in honey of the brood frames immediately after the feeding and decreased over time to low concentrations at the end of the year. A high content of S. cerevisiae cells were also found in the honey from supers of the spring harvest. Observed S. cerevisiae cells were not able to multiply in a high-sugar environment, such as honey, and their viability decreased rapidly after addition to the honey. The screening of 200 Swiss honeys revealed the presence of S. cerevisiae in 4.5% of the samples, as determined by microscopy and qPCR. Finally, the method described here may indicate an unwanted sucrose addition to honey through bee-feeding. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production

    PubMed Central

    Vigentini, Ileana; Gebbia, Marinella; Belotti, Alessandra; Foschino, Roberto; Roth, Frederick P.

    2017-01-01

    An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial “starter” strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8–12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts. PMID:29163459

  15. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  16. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  17. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  18. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dried yeasts. 172.896 Section 172.896 Food and... Multipurpose Additives § 172.896 Dried yeasts. Dried yeast (Saccharomyces cerevisiae and Saccharomyces fragilis) and dried torula yeast (Candida utilis) may be safely used in food provided the total folic acid...

  19. [Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

    PubMed

    Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

    2016-01-01

    A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

  20. Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

    PubMed Central

    Eremenko, Ekaterina; Ben-Zvi, Anat; Morozova-Roche, Ludmilla A.; Raveh, Dina

    2013-01-01

    Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery. PMID:23483999

  1. Application of anhydrobiosis and dehydration of yeasts for non-conventional biotechnological goals.

    PubMed

    Rapoport, Alexander; Turchetti, Benedetta; Buzzini, Pietro

    2016-06-01

    Dehydration of yeast cells causes them to enter a state of anhydrobiosis in which their metabolism is temporarily and reversibly suspended. This unique state among organisms is currently used in the production of active dry yeasts, mainly used in baking and winemaking. In recent decades non-conventional applications of yeast dehydration have been proposed for various modern biotechnologies. This mini-review briefly summarises current information on the application of dry yeasts in traditional and innovative fields. It has been shown that dry yeast preparations can be used for the efficient protection, purification and bioremediation of the environment from heavy metals. The high sorption activity of dehydrated yeasts can be used as an interesting tool in winemaking due to their effects on quality and taste. Dry yeasts are also used in agricultural animal feed. Another interesting application of yeast dehydration is as an additional stage in new methods for the stable immobilisation of microorganisms, especially in cases when biotechnologically important strains have no affinity with the carrier. Such immobilisation methods also provide a new approach for the successful conservation of yeast strains that are very sensitive to dehydration. In addition, the application of dehydration procedures opens up new possibilities for the use of yeast as a model system. Separate sections of this review also discuss possible uses of dry yeasts in biocontrol, bioprotection and biotransformations, in analytical methods as well as in some other areas.

  2. A Method of Visualizing Three-Dimensional Distribution of Yeast in Bread Dough

    NASA Astrophysics Data System (ADS)

    Maeda, Tatsurou; Do, Gab-Soo; Sugiyama, Junichi; Oguchi, Kosei; Shiraga, Seizaburou; Ueda, Mitsuyoshi; Takeya, Koji; Endo, Shigeru

    A novel technique was developed to monitor the change in three-dimensional (3D) distribution of yeast in frozen bread dough samples in accordance with the progress of mixing process. Application of a surface engineering technology allowed the identification of yeast in bread dough by bonding EGFP (Enhanced Green Fluorescent Protein) to the surface of yeast cells. The fluorescent yeast (a biomarker) was recognized as bright spots at the wavelength of 520 nm. A Micro-Slicer Image Processing System (MSIPS) with a fluorescence microscope was utilized to acquire cross-sectional images of frozen dough samples sliced at intervals of 1 μm. A set of successive two-dimensional images was reconstructed to analyze 3D distribution of yeast. Samples were taken from each of four normal mixing stages (i.e., pick up, clean up, development, and final stages) and also from over mixing stage. In the pick up stage yeast distribution was uneven with local areas of dense yeast. As the mixing progressed from clean up to final stages, the yeast became more evenly distributed throughout the dough sample. However, the uniformity in yeast distribution was lost in the over mixing stage possibly due to the breakdown of gluten structure within the dough sample.

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

    DOEpatents

    Young, Travis; Schultz, Peter G.

    2017-08-15

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

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

    DOEpatents

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

    2014-02-11

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

  5. New insights into the capacity of commercial wine yeasts to grow on sparkling wine media. Factor screening for improving wine yeast selection.

    PubMed

    Borrull, Anna; Poblet, Montse; Rozès, Nicolas

    2015-06-01

    During the production of sparkling wine, wine yeasts are subjected to many stress factors apart from ethanol, which lead to the need to achieve their acclimation in line with various industrial protocols. In the present work, 44 commercial wine Saccharomyces cerevisiae strains and one laboratory strain (BY4742) were firstly subjected to the influence of increasing concentrations of ethanol to cluster the yeasts using discriminant function analysis. Afterwards, non-inhibitory concentration (NIC) and minimum inhibitory concentration (MIC) were estimated, revealing some differences between 24 of these strains. Meanwhile, this study confirms the negative synergistic effect of low pH with ethanol on the maximum specific growth rate (μmax) and lag phase time. Moreover, a negative effect of increasing levels of glycerol in the growth medium was observed. Interestingly enough, an interactive positive effect was found between cysteine and medium-chain fatty acids (MCFA). While cysteine did not have a really significant effect in comparison to the control, it was able to restore the damage caused by MCFA, making the growth rate of cells recover and even reducing the formation of reactive oxygen species. Adequate culture aeration is also crucial for the composition of the cell fatty acid. The final results showed that few differences were observed between NIC and MIC estimations with respect to cells pre-cultured in the presence or absence of oxygen. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

    PubMed

    Yukawa, Masashi; Yamauchi, Tomoaki; Kurisawa, Naoaki; Ahmed, Shakil; Kimura, Ken-Ichi; Toda, Takashi

    2018-07-01

    Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

    PubMed

    Chen, Xianzhong

    2017-03-04

    The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

  8. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance

    PubMed Central

    Chen, Xianzhong

    2017-01-01

    ABSTRACT The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed. PMID:27459271

  9. Systematic exploration of essential yeast gene function with temperature-sensitive mutants

    PubMed Central

    Li, Zhijian; Vizeacoumar, Franco J; Bahr, Sondra; Li, Jingjing; Warringer, Jonas; Vizeacoumar, Frederick S; Min, Renqiang; VanderSluis, Benjamin; Bellay, Jeremy; DeVit, Michael; Fleming, James A; Stephens, Andrew; Haase, Julian; Lin, Zhen-Yuan; Baryshnikova, Anastasia; Lu, Hong; Yan, Zhun; Jin, Ke; Barker, Sarah; Datti, Alessandro; Giaever, Guri; Nislow, Corey; Bulawa, Chris; Myers, Chad L; Costanzo, Michael; Gingras, Anne-Claude; Zhang, Zhaolei; Blomberg, Anders; Bloom, Kerry; Andrews, Brenda; Boone, Charles

    2012-01-01

    Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes. PMID:21441928

  10. Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

    PubMed Central

    Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

    1987-01-01

    A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer. PMID:16347298

  11. Yeast flocculation: New story in fuel ethanol production.

    PubMed

    Zhao, X Q; Bai, F W

    2009-01-01

    Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.

  12. Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

    PubMed Central

    Smith, Ida M.; Christensen, Jeffrey E.; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  13. Brewing characteristics of piezosensitive sake yeasts

    NASA Astrophysics Data System (ADS)

    Nomura, Kazuki; Hoshino, Hirofumi; Igoshi, Kazuaki; Onozuka, Haruka; Tanaka, Erika; Hayashi, Mayumi; Yamazaki, Harutake; Takaku, Hiroaki; Iguchi, Akinori; Shigematsu, Toru

    2018-04-01

    Application of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Saccharomyces cerevisiae used for Japanese rice wine (sake) brewing shows high tolerance to HHP. Therefore, we aimed to generate pressure-sensitive (piezosensitive) sake yeast strains by mating sake with piezosensitive yeast strains to establish an HHP fermentation regulation technology and extend the shelf life of fermented foods. The results of phenotypic analyses showed that the generated yeast strains were piezosensitive and exhibited similar fermentation ability compared with the original sake yeast strain. In addition, primary properties of sake brewed using these strains, such as ethanol concentration, sake meter value and sake flavor compounds, were almost equivalent to those obtained using the sake yeast strain. These results suggest that the piezosensitive strains exhibit brewing characteristics essentially equivalent to those of the sake yeast strain.

  14. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

    PubMed

    Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

    2015-05-01

    Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

  15. Effect of Vibration on Retention Characteristics of Screen Acquisition Systems

    NASA Technical Reports Server (NTRS)

    Tegart, J. R.; Park, A. C.

    1977-01-01

    An analytical and experimental investigation of the effect of vibration on the retention characteristics of screen acquisition systems was performed. The functioning of surface tension devices using fine-mesh screens requires that the pressure differential acting on the screen be less than its pressure retention capability. When exceeded, screen breakdown will occur and gas-free expulsion of propellant will no longer be possible. An analytical approach to predicting the effect of vibration was developed. This approach considers the transmission of the vibration to the screens of the device and the coupling of the liquid and the screen in establishing the screen response. A method of evaluating the transient response of the gas/liquid interface within the screen was also developed.

  16. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and dried cell walls of the yeast...

  17. Smart Screening System (S3) In Taconite Processing

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

    Daryoush Allaei; Ryan Wartman; David Tarnowski

    2006-03-01

    key components include smart motor and associated electronics, resonators, and supporting structural elements. It is shown that the smart motors have an acceptable life and performance. Resonator (or motion amplifier) designs are selected based on the final system requirement and vibration characteristics. All the components for a fully functional prototype are fabricated. The development program is on schedule. The last semi-annual report described the completion of the design refinement phase. This phase resulted in a Smart Screen design that meets performance targets both in the dry condition and with taconite slurry flow using PZT motors. This system was successfully demonstrated for the DOE and partner companies at the Coleraine Mineral Research Laboratory in Coleraine, Minnesota. Since then, the fabrication of the dry application prototype (incorporating an electromagnetic drive mechanism and a new deblinding concept) has been completed and successfully tested at QRDC's lab.« less

  18. The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium.

    PubMed

    Smith, Brendan D; Divol, Benoit

    2018-08-01

    The wine matrix contains limited carbon compounds to sustain microbial life. Brettanomyces bruxellensis is one of very few yeast species that has adapted to this environment. Indeed, the presence of growth-inhibiting compounds and conditions do not prevent its proliferation. Literature regarding the nutritional requirements of this yeast is surprisingly poor, given the observation that B. bruxellensis produces biomass with apparently less nutrients than other yeasts. In this study, various carbon sources were screened in a synthetic wine medium, under anaerobic and semi-aerobic growth conditions, in order to determine which compounds B. bruxellensis assimilates. Slight differences were observed between strains but overall, B. bruxellensis produced biomass from limited nutrients consumed in a specific order regardless of the oxygen conditions. Upon initial consumption of the simple sugars, B. bruxellensis was able to remain viable, by concurrently utilising ethanol (only in the presence of oxygen) and malic acid. Although initially beneficial, oxygen was found detrimental in the long term. Formation of volatile phenols occurred during the consumption of the sugars but not as a mechanism to help correct the redox imbalance. The study confirms that B. bruxellensis is able to survive using limited amount of nutrients, making this yeast a challenge for winemakers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Analyzing and Understanding Lipids of Yeast: A Challenging Endeavor.

    PubMed

    Kohlwein, Sepp D

    2017-05-01

    Lipids are essential biomolecules with diverse biological functions, ranging from building blocks for all biological membranes to energy substrates, signaling molecules, and protein modifiers. Despite advances in lipid analytics by mass spectrometry, the extraction and quantitative analysis of the diverse classes of lipids are still an experimental challenge. Yeast is a model organism that provides several advantages for studying lipid metabolism, because most biosynthetic pathways are well described and a great deal of information is available on the regulatory mechanisms that control lipid homeostasis. In addition, the composition of yeast lipids is much less complex than that of mammalian lipids, making yeast an excellent reference system for studying lipid-associated cell functions. © 2017 Cold Spring Harbor Laboratory Press.

  20. Bio-prospecting of distillery yeasts as bio-control and bio-remediation agents.

    PubMed

    Ubeda, Juan F; Maldonado, María; Briones, Ana I; Francisco, J Fernández; González, Francisco J

    2014-05-01

    This work constitutes a preliminary study in which the capacity of non-Saccharomyces yeasts isolated from ancient distilleries as bio-control agents against moulds and in the treatment of waste waters contaminated by heavy metals-i.e. bio-remediation-is shown. In the first control assays, antagonist effect between non-Saccharomyces yeasts, their extracts and supernatants against some moulds, analysing the plausible (not exhaustive) involved factors were qualitatively verified. In addition, two enzymatic degrading properties of cell wall plant polymers, quitinolitic and pectinolitic, were screened. Finally, their use as agents of bio-remediation of three heavy metals (cadmium, chromium and lead) was analysed semi-quantitatively. The results showed that all isolates belonging to Pichia species effectively inhibited all moulds assayed. Moreover, P. kudriavzevii is a good candidate for both bio-control and bio-remediation because it inhibited moulds and accumulated the major proportion of the three tested metals.

  1. 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

  2. Opportunistic Pathogenic Yeasts

    NASA Astrophysics Data System (ADS)

    Banerjee, Uma

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

  3. Characterization of the nitrogen compounds released during yeast autolysis in a model wine system.

    PubMed

    Martínez-Rodríguez, A J; Polo, M C

    2000-04-01

    The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.

  4. Screening of some essential oils against Trichosporon species.

    PubMed

    Uniyal, Veena; Saxena, Seema; Bhatt, R P

    2013-01-01

    White Piedra is a superficial mycoses characterized by nodules on the hair shaft, caused by the basidiomycetous yeast Trichosporon species. In this study 25 essential oils were extracted and screened against two Trichosporon species i.e. Trichosporon asahii and Trichosporon cutaneum. Both these fungi procured from MTCC Chandigarh were maintained on yeast malt agar plates and tubes at 25 degrees C. Two screening methods viz., agar well diffusion assay and minimum inhibitory concentration were adopted for the study. The results showed that the maximum anti-yeast activity against T. asahii and T. cutaneum was demonstrated by oil of Mentha piperita showing full inhibition of both the fungi, Melaleuca alternifolia with an inhibition zone of 45 and 40 mm, Cymbopogon winterians with inhibition zone of 45 and 45 mm and Cymbopogon flexuosus with 35 and 30 mm inhibition zones. The oil of Trachyspermum ammi exhibited 10 and 20 mm, Abelmoschus moschatus exhibited 30 and 20 mm, Salvia sclarea showed 20 and 18 mm and Jasminum officinale exhibited 25 and 15 mm inhibition zones showing moderate activity. The oil of Cyperus scariosus, Pogostemon patchouli and Rosa damascene showed no inhibition zone against both the fungi while Vetiveria zizanoides exhibited no inhibition in case of T. asahii and inhibition zone of 10 mm in case of T. cutaneum demonstrating comparatively low activity against both the fungi. These results support that the essential oils can be used to cure superficial mycoses and these oils may have significant role as pharmaceuticals and preservatives.

  5. 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

  6. 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

  7. Yeast Prions and Human Prion-like Proteins: Sequence Features and Prediction Methods

    PubMed Central

    Cascarina, Sean; Ross, Eric D.

    2014-01-01

    Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis (ALS). This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms. PMID:24390581

  8. Yeast prions and human prion-like proteins: sequence features and prediction methods.

    PubMed

    Cascarina, Sean M; Ross, Eric D

    2014-06-01

    Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.

  9. 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)

  10. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  11. 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.

  12. Automated multiplex genome-scale engineering in yeast

    PubMed Central

    Si, Tong; Chao, Ran; Min, Yuhao; Wu, Yuying; Ren, Wen; Zhao, Huimin

    2017-01-01

    Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast. PMID:28469255

  13. Continuous immobilized yeast reactor system for complete beer fermentation using spent grains and corncobs as carrier materials.

    PubMed

    Brányik, Tomás; Silva, Daniel P; Vicente, António A; Lehnert, Radek; e Silva, João B Almeida; Dostálek, Pavel; Teixeira, José A

    2006-12-01

    Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packed-bed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.

  14. Effect of transient liquid flow on retention characteristics of screen acquisition systems. [design of Space Shuttle feed system

    NASA Technical Reports Server (NTRS)

    Cady, E. C.

    1977-01-01

    A design analysis, is developed based on experimental data, to predict the effects of transient flow and pressure surges (caused either by valve or pump operation, or by boiling of liquids in warm lines) on the retention performance of screen acquisition systems. A survey of screen liquid acquisition system applications was performed to determine appropriate system environment and classification. A screen model was developed which assumed that the screen device was a uniformly distributed composite orthotropic structure, and which accounted for liquid inflow/outflow, gas ingestion quality, screen stress, and liquid spill. A series of 177 tests using 13 specimens (5 screen meshes, 4 screen device construction/backup methods, and 2 orientations) with three test fluids (isopropyl alcohol, Freon 114, and LH2) provided data which verified important features of the screen model and resulted in a design tool which could accurately predict the transient startup performance acquisition devices.

  15. 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.

  16. 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

  17. 'Yeast mail': a novel Saccharomyces application (NSA) to encrypt messages.

    PubMed

    Rosemeyer, Helmut; Paululat, Achim; Heinisch, Jürgen J

    2014-09-01

    The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)-based, one-step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  18. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

    PubMed

    Skjoedt, Mette L; Snoek, Tim; Kildegaard, Kanchana R; Arsovska, Dushica; Eichenberger, Michael; Goedecke, Tobias J; Rajkumar, Arun S; Zhang, Jie; Kristensen, Mette; Lehka, Beata J; Siedler, Solvej; Borodina, Irina; Jensen, Michael K; Keasling, Jay D

    2016-11-01

    Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

  19. Yeast strains as potential aroma enhancers in dry fermented sausages.

    PubMed

    Flores, Mónica; Corral, Sara; Cano-García, Liliana; Salvador, Ana; Belloch, Carmela

    2015-11-06

    Actual healthy trends produce changes in the sensory characteristics of dry fermented sausages therefore, new strategies are needed to enhance their aroma. In particular, a reduction in the aroma characteristics was observed in reduced fat and salt dry sausages. In terms of aroma enhancing, generally coagulase-negative cocci were selected as the most important group from the endogenous microbiota in the production of flavour compounds. Among the volatile compounds analysed in dry sausages, ester compounds contribute to fruity aroma notes associated with high acceptance of traditional dry sausages. However, the origin of ester compounds in traditional dry sausages can be due to other microorganisms as lactic acid bacteria, yeast and moulds. Yeast contribution in dry fermented sausages was investigated with opposite results attributed to low yeast survival or low activity during processing. Generally, they affect sausage colour and flavour by their oxygen-scavenging and lipolytic activities in addition to, their ability to catabolize fermentation products such as lactate increasing the pH and contributing to less tangy and more aromatic sausages. Recently, the isolation and characterization of yeast from traditional dry fermented sausages made possible the selection of those with ability to produce aroma active compounds. Molecular methods were used for genetic typing of the isolated yeasts whereas their ability to produce aroma compounds was tested in different systems such as in culture media, in model systems and finally on dry fermented sausages. The results revealed that the appropriate selection of yeast strains with aroma potential may be used to improve the sensory characteristics of reformulated fermented sausages. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Identification of the fitness determinants of budding yeast on a natural substrate

    PubMed Central

    Filteau, Marie; Charron, Guillaume; Landry, Christian R

    2017-01-01

    The budding yeasts are prime models in genomics and cell biology, but the ecological factors that determine their success in non-human-associated habitats is poorly understood. In North America Saccharomyces yeasts are present on the bark of deciduous trees, where they feed on bark and sap exudates. In the North East, Saccharomyces paradoxus is found on maples, which makes maple sap a natural substrate for this species. We measured growth rates of S. paradoxus natural isolates on maple sap and found variation along a geographical gradient not explained by the inherent variation observed under optimal laboratory conditions. We used a functional genomic screen to reveal the ecologically relevant genes and conditions required for optimal growth in this substrate. We found that the allantoin degradation pathway is required for optimal growth in maple sap, in particular genes necessary for allantoate utilization, which we demonstrate is the major nitrogen source available to yeast in this environment. Growth with allantoin or allantoate as the sole nitrogen source recapitulated the variation in growth rates in maple sap among strains. We also show that two lineages of S. paradoxus display different life-history traits on allantoin and allantoate media, highlighting the ecological relevance of this pathway. PMID:27935595

  1. Identification of the fitness determinants of budding yeast on a natural substrate.

    PubMed

    Filteau, Marie; Charron, Guillaume; Landry, Christian R

    2017-04-01

    The budding yeasts are prime models in genomics and cell biology, but the ecological factors that determine their success in non-human-associated habitats is poorly understood. In North America Saccharomyces yeasts are present on the bark of deciduous trees, where they feed on bark and sap exudates. In the North East, Saccharomyces paradoxus is found on maples, which makes maple sap a natural substrate for this species. We measured growth rates of S. paradoxus natural isolates on maple sap and found variation along a geographical gradient not explained by the inherent variation observed under optimal laboratory conditions. We used a functional genomic screen to reveal the ecologically relevant genes and conditions required for optimal growth in this substrate. We found that the allantoin degradation pathway is required for optimal growth in maple sap, in particular genes necessary for allantoate utilization, which we demonstrate is the major nitrogen source available to yeast in this environment. Growth with allantoin or allantoate as the sole nitrogen source recapitulated the variation in growth rates in maple sap among strains. We also show that two lineages of S. paradoxus display different life-history traits on allantoin and allantoate media, highlighting the ecological relevance of this pathway.

  2. Functional Coupling of a Nematode Chemoreceptor to the Yeast Pheromone Response Pathway

    PubMed Central

    Tehseen, Muhammad; Dumancic, Mira; Briggs, Lyndall; Wang, Jian; Berna, Amalia; Anderson, Alisha; Trowell, Stephen

    2014-01-01

    Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) triple mutant (“Cyb”) with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors. PMID:25415379

  3. 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

  4. Automated screening of propulsion system test data by neural networks, phase 1

    NASA Technical Reports Server (NTRS)

    Hoyt, W. Andes; Whitehead, Bruce A.

    1992-01-01

    The evaluation of propulsion system test and flight performance data involves reviewing an extremely large volume of sensor data generated by each test. An automated system that screens large volumes of data and identifies propulsion system parameters which appear unusual or anomalous will increase the productivity of data analysis. Data analysts may then focus on a smaller subset of anomalous data for further evaluation of propulsion system tests. Such an automated data screening system would give NASA the benefit of a reduction in the manpower and time required to complete a propulsion system data evaluation. A phase 1 effort to develop a prototype data screening system is reported. Neural networks will detect anomalies based on nominal propulsion system data only. It appears that a reasonable goal for an operational system would be to screen out 95 pct. of the nominal data, leaving less than 5 pct. needing further analysis by human experts.

  5. Identification of a novel pathway involving a GATA transcription factor in yeast and possibly plant Zn uptake and homeostasis

    USDA-ARS?s Scientific Manuscript database

    To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Nocceae caerulescens, was screened for its ability to restore growth under Zn limitin...

  6. Evolution of the hemiascomycete yeasts: on life styles and the importance of inbreeding.

    PubMed

    Knop, Michael

    2006-07-01

    The term 'breeding system' is used to describe the morphological and behavioural aspects of the sexual life cycle of a species. The yeast breeding system provides three alternatives that enable hapoids to return to the diploid state that is necessary for meiosis: mating of unrelated haploids (amphimixis), mating between spores from the same tetrad (intratetrad mating, automixis) and mother daughter mating upon mating type switching (haplo-selfing). The frequency of specific mating events affects the level of heterozygosity present in individuals and the genetic diversity of populations. This review discusses the reproductive strategies of yeasts, in particular S. cerevisiae (Bakers' or budding yeast). Emphasis is put on intratetrad mating, its implication for diversity, and how the particular genome structure could have evolved to ensure the preservation of a high degree of heterozygosity in conjunction with frequent intratetrad matings. I also discuss how the ability of yeast to control the number of spores that are formed accounts for high intratetrad mating rates and for enhanced transmission of genomic variation. I extend the discussion to natural genetic variation and propose that a high level of plasticity is inherent in the yeast breeding system, which may allow variation of the breeding behaviour in accordance with the needs imposed by the environment. (c) 2006 Wiley Periodicals, Inc.

  7. Systemic changes and adverse effects induced by retinopathy of prematurity screening.

    PubMed

    Jiang, Jing-Bo; Zhang, Zhi-Wei; Zhang, Jia-Wen; Wang, Yan-Li; Nie, Chuan; Luo, Xian-Qiong

    2016-01-01

    To estimate the potential systemic events during and after retinopathy of prematurity (ROP) screening. A prospective and descriptive designed study was conducted to detect the physiologic and pathological changes 24h before, during, and 72h after ROP screening. Control blood pressure (BP), saturation, pulse rate, and body temperature were routinely taken at various time internals before and after screening. Adverse effects pertain to cardiovascular system, respiratory system, gastric system, urinary system and nervous system were retrospect 0-72h after ROP screening at a 24-hour interval. Totally 1254 prematurity babies receiving ROP screening during Jan. 1(st) 2013 to Dec. 31(th) 2013 were enrolled in our survey. Compared to control vital sign data taken before the examination, there was a fluctuation in the diastolic BP with the increased 3.03 mm Hg (P=0.04) after 3 doses of mydriatic drops. Immediately after the examination, there was a further 12.64 mm Hg (P<0.01) increase in systolic BP and a 7.24 mm Hg (P<0.01) in diastolic BP. The mean pulse rate during examination was 22.4 bpm (P<0.01) higher than the 133.3±9.0 bpm control level. The oxygen saturation shared an average drop of 5% (P<0.01) during screening. In prematurity with postconceptional age less than 31wk, the incidence of apnea (23.5%), necrotizing enterocolitis (NEC) (8.7%), gastric residual (25.4%) and upper digestive tract hemorrhage (6.4%) also demonstrated a significant rise (P<0.01). In our study sample, ROP screening was associated with NEC, gastric residual and upper digestive tract hemorrhage. These gastrointestinal side effects, along with breath activity pattern change and vital signs indicators fluctuation, may be results of additional stress responses.

  8. 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.

  9. Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts.

    PubMed

    Tan, Amelia W J; Lee, Pin-Rou; Seow, Yi-Xin; Ong, Peter K C; Liu, Shao-Quan

    2012-08-01

    Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.

  10. Potential of plant genetic systems for monitoring and screening mutagens

    PubMed Central

    Nilan, R. A.

    1978-01-01

    Plants have too long been ignored as useful screening and monitoring systems of environmental mutagens. However, there are about a dozen reliable, some even unique, plant genetic systems that can increase the scope and effectiveness of chemical and physical mutagen screening and monitoring procedures. Some of these should be included in the Tier II tests. Moreover, plants are the only systems now in use as monitors of genetic effects caused by polluted atmosphere and water and by pesticides. There are several major advantages of the plant test systems which relate to their reproductive nature, easy culture and growth habits that should be considered in mutagen screening and monitoring. In addition to these advantages, the major plant test systems exhibit numerous genetic and chromosome changes for determining the effects of mutagens. Some of these have not yet been detected in other nonmammalian and mammalian test systems, but probably occur in the human organism. Plants have played major roles in various aspects of mutagenesis research, primarily in mutagen screening (detection and verification of mutagenic activity), mutagen monitoring, and determining mutagen effects and mechanisms of mutagen action. They have played lesser roles in quantification of mutagenic activity and understanding the nature of induced mutations. Mutagen monitoring with plants, especially in situ on land or in water, will help determine potential genetic hazards of air and water pollutants and protect the genetic purity of crop plants and the purity of the food supply. The Tradescantia stamen-hair system is used in a mobile laboratory for determining the genetic effects of industrial and automobile pollution in a number of sites in the U.S.A. The fern is employed for monitoring genetic effects of water pollution in the Eastern states. The maize pollen system and certain weeds have monitored genetic effects of pesticides. Several other systems that have considerable value and should be

  11. Yeasts as distinct life forms of fungi

    USDA-ARS?s Scientific Manuscript database

    This review describes all presently recognized genera of the Ascomycete yeasts (Saccharomycotina, budding yeasts, and the Taphrinomycotina, fission yeasts and related) as well as all currently recognized genera of the Basidiomycete yeasts. This update will be the lead chapter for a book entitled “Ye...

  12. Carbonation acceleration of calcium hydroxide nanoparticles: induced by yeast fermentation

    NASA Astrophysics Data System (ADS)

    Lopez-Arce, Paula; Zornoza-Indart, Ainara

    2015-09-01

    Carbonation of Ca(OH)2 nanoparticles and consolidation of limestone are accelerated by high humidity and a yeast fermentation system that supplies a saturated atmosphere on CO2, H2O vapor and ethanol during 28 days. Nanoparticles were analyzed by X-ray diffraction and differential thermal analyses with thermogravimetry. Spectrophotometry, scanning electron microscopy analyses, and hydric and mechanical tests were also performed in stones specimens. Samples exposed to the yeast environment achieve 100 % relative CaCO3 yield, whereas at high humidity but without the yeast and under laboratory environment, relative yields of 95 % CaCO3 and 15 % CaCO3 are, respectively, reached, with white crusts and glazing left on the stone surfaces when the nanoparticles are applied at a concentration of 25 g/l. The largest increase in the drilling resistance and surface hardness values with slight increase in the capillarity absorption and desorption coefficients and with lesser stone color changes are produced at a concentration of 5 g/l, in the yeast system environment. This especially happens in stone specimens initially with bimodal pore size distributions, more amounts of pores with diameters between 0.1 and 1 µm, higher open porosity values and faster capillary coefficients. An inexpensive and reliable method based on water and yeast-sugar solution is presented to speed up carbonation of Ca(OH)2 nanoparticles used as a consolidating product to improve the mechanical properties of decayed limestone from archaeological and architectural heritage.

  13. Evolutionary History of Ascomyceteous Yeasts

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

    Haridas, Sajeet; Riley, Robert; Salamov, Asaf

    2014-06-06

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with alternative nuclear codon usage where CUG encodes serine instead of leucine are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with amore » large fraction of single exon genes with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.« less

  14. Eighteen new oleaginous yeast species.

    PubMed

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

    2016-07-01

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

  15. Teaching nutritional biochemistry: an experimental approach using yeast.

    PubMed

    Alonso, Manuel; Stella, Carlos A

    2012-12-01

    In this report, we present a practical approach to teaching several topics in nutrition to science students at the high school and college freshmen levels. This approach uses baker's yeast (Saccharomyces cerevisiae) as a biological system model. The diameters of yeast colonies, which vary according to the nutrients present in the medium, can be observed, compared, and used to teach metabolic requirements. The experiments described in this report show simple macroscopic evidence of submicroscopic nutritional events. This can serve as a useful base for an analogy of heterotrophic human cell nutrition.

  16. Down-regulation of intestinal epithelial innate response by probiotic yeasts isolated from kefir.

    PubMed

    Romanin, David; Serradell, María; González Maciel, Dolores; Lausada, Natalia; Garrote, Graciela L; Rumbo, Martín

    2010-06-15

    Kefir is obtained by milk fermentation with a complex microbial population included in a matrix of polysaccharide and proteins. Several health-promoting activities has been attributed to kefir consumption. The aim of this study was to select microorganisms from kefir able to down-regulate intestinal epithelial innate response and further characterize this activity. Caco-2 cells stably transfected with a human CCL20 promoter luciferase reporter were used to screen a collection of 24 yeast and 23 bacterial strains isolated from kefir. The Toll-like receptor 5 agonist, flagellin was used to activate the reporter cells, while pre-incubation with the selected strains was tested to identify strains with the capacity to inhibit cell activation. In this system, 21 yeast strains from the genera Saccharomyces, Kluyveromyces and Issatchenkia inhibited almost 100% of the flagellin-dependent activation, whereas only some lactobacilli strains showed a partial effect. K. marxianus CIDCA 8154 was selected for further characterization. Inhibitory activity was confirmed at transcriptional level on Caco-2/TC-7 and HT-29 cells upon flagellin stimulation. A similar effect was observed using other pro-inflammatory stimulation such as IL-1beta and TNF-alpha. Pre-incubation with yeasts induced a down-regulation of NF-kappaB signalling in epithelial cells in vitro, as well as expression of other pro-inflammatory chemokines such as CXCL8 and CXCL2. Furthermore, modulation of CCL20 mRNA expression upon flagellin stimulation was evidenced in vivo, in a mouse ligated intestinal loop model. Results indicate kefir contains microorganisms able to abolish the intestinal epithelial inflammatory response that could explain some of the properties attributed to this fermented milk. Copyright 2010 Elsevier B.V. All rights reserved.

  17. Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

    DOE PAGES

    Sun, Yue; Ban, Bhupal; Bradbury, Andrew; ...

    2016-08-29

    The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

  18. Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

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

    Sun, Yue; Ban, Bhupal; Bradbury, Andrew

    The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

  19. Screening of yeasts as probiotic based on capacities to colonize the gastrointestinal tract and to protect against enteropathogen challenge in mice.

    PubMed

    Martins, Flariano S; Nardi, Regina M D; Arantes, Rosa M E; Rosa, Carlos A; Neves, Maria J; Nicoli, Jacques R

    2005-04-01

    Probiotics are defined as viable microorganisms that exhibit a beneficial effect on the host's health when they are ingested. Two important criteria are used for selection of probiotic microorganisms: they must be able to survive in the gastrointestinal environment and to present at least one beneficial function (colonization resistance, immunomodulation or nutritional contribution). Generally, in vitro assays demonstrating these properties were used to select probiotics but it is unclear if the data can be extrapolated to in vivo conditions. In the present work, twelve Saccharomyces cerevisiae strains isolated from different environments (insect association, tropical fruit, cheese and "aguardente" production) and pre-selected for in vitro resistance to simulated gastrointestinal conditions were inoculated in germ-free mice to evaluate their real capacity to colonize the mammal digestive tract. Using these data, one of the yeasts (S. cerevisiae 905) was selected and tested in gnotobiotic (GN) and conventional (CV) mice for its capacity to protect against oral challenge with two enteropathogenic bacteria (Salmonella Typhimurium and Clostridium difficile). The yeast reached populational levels potentially functional in the gastrointestinal portions where the enteropathogens tested act. No antagonism against either pathogenic bacterium by the yeast was observed in the digestive tract of GN mice but, after challenge with S. Typhimurium, mortality was lower and liver tissue was better preserved in CV animals treated with the yeast when compared with a control group (p<0.05). Histopathological results of intestines showed that the yeast also presented a good protective effect against oral challenge with C. difficile in GN mice (p<0.05). In conclusion, among the 12 S. cerevisiae tested, strain 905 showed the best characteristics to be used as a probiotic as demonstrated by survival capacity in the gastrointestinal tract and protective effect of animals during experimental

  20. Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

    PubMed

    Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

    2009-05-01

    Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

  1. Oral yeast colonization throughout pregnancy

    PubMed Central

    Rio, Rute; Simões-Silva, Liliana; Garro, Sofia; Silva, Mário-Jorge; Azevedo, Álvaro

    2017-01-01

    Background Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. Material and Methods The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Results Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Conclusions Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment. Key words:Oral yeast, fungi, pregnancy, saliva pH. PMID:28160578

  2. Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

    PubMed

    Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

    2017-08-01

    Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

  3. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Yeasts isolated from Algerian infants's feces revealed a burden of Candida albicans species, non-albicans Candida species and Saccharomyces cerevisiae.

    PubMed

    Seddik, Hamza Ait; Ceugniez, Alexandre; Bendali, Farida; Cudennec, Benoit; Drider, Djamel

    2016-01-01

    This study aimed at showing the yeast diversity in feces of Algerian infants, aged between 1 and 24 months, hospitalized at Bejaia hospital (northeast side of the country). Thus, 20 colonies with yeast characteristics were isolated and identified using biochemical (ID32C Api system) and molecular (sequencing of ITS1-5.8S-ITS2 region) methods. Almost all colonies isolated (19 strains) were identified as Candida spp., with predominance of Candida albicans species, and one strain was identified as Saccharomyces cerevisiae. Screening of strains with inhibitory activities unveiled the potential of Candida parapsilosis P48L1 and Candida albicans P51L1 to inhibit the growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Further studies performed with these two Candida strains revealed their susceptibility to clinically used antifungal compounds and were then characterized for their cytotoxicity and hemolytic properties. On the other hand, Saccharomyces cerevisiae P9L1 isolated as well in this study was shown to be devoid of antagonism but resulted safe and overall usable as probiotic.

  5. Yarrowia lipolytica: a model yeast for citric acid production.

    PubMed

    Cavallo, Ema; Charreau, Hernán; Cerrutti, Patricia; Foresti, María Laura

    2017-12-01

    Every year more than 2 million tons of citric acid (CA) are produced around the world for industrial uses. Although initially extracted from citrus, the low profitability of the process and the increasing demand soon stimulated the search for more efficient methods to produce CA. Currently, most world CA demand (99%) is satisfied by fermentations with microorganisms, especially filamentous fungi and yeasts. CA production with yeasts has certain advantages over molds (e.g. higher productivity and easier cultivation), which in the last two decades have triggered a clear increase in publications and patents devoted to the use of yeasts in this field. Yarrowia lipolytica has become a model yeast that proved to be successful in different production systems. Considering the current interest evidenced in the literature, the most significant information on CA production using Y. lipolytica is summarized. The relevance on CA yields of key factors such as strains, media formulation, environmental conditions and production regimes is thoroughly discussed, with particular focus on increasing CA productivity. Besides, the possibility of tuning the mentioned variables to reduce concomitant isocitric acid production-the biggest disadvantage of using yeasts-is analyzed. Available methods for CA purification/quantification are also discussed. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Applying operations research to optimize a novel population management system for cancer screening.

    PubMed

    Zai, Adrian H; Kim, Seokjin; Kamis, Arnold; Hung, Ken; Ronquillo, Jeremiah G; Chueh, Henry C; Atlas, Steven J

    2014-02-01

    To optimize a new visit-independent, population-based cancer screening system (TopCare) by using operations research techniques to simulate changes in patient outreach staffing levels (delegates, navigators), modifications to user workflow within the information technology (IT) system, and changes in cancer screening recommendations. TopCare was modeled as a multiserver, multiphase queueing system. Simulation experiments implemented the queueing network model following a next-event time-advance mechanism, in which systematic adjustments were made to staffing levels, IT workflow settings, and cancer screening frequency in order to assess their impact on overdue screenings per patient. TopCare reduced the average number of overdue screenings per patient from 1.17 at inception to 0.86 during simulation to 0.23 at steady state. Increases in the workforce improved the effectiveness of TopCare. In particular, increasing the delegate or navigator staff level by one person improved screening completion rates by 1.3% or 12.2%, respectively. In contrast, changes in the amount of time a patient entry stays on delegate and navigator lists had little impact on overdue screenings. Finally, lengthening the screening interval increased efficiency within TopCare by decreasing overdue screenings at the patient level, resulting in a smaller number of overdue patients needing delegates for screening and a higher fraction of screenings completed by delegates. Simulating the impact of changes in staffing, system parameters, and clinical inputs on the effectiveness and efficiency of care can inform the allocation of limited resources in population management.

  7. Game dynamic model for yeast development.

    PubMed

    Huang, Yuanyuan; Wu, Zhijun

    2012-07-01

    Game theoretic models, along with replicator equations, have been applied successfully to the study of evolution of populations of competing species, including the growth of a population, the reaching of the population to an equilibrium state, and the evolutionary stability of the state. In this paper, we analyze a game model proposed by Gore et al. (Nature 456:253-256, 2009) in their recent study on the co-development of two mixed yeast strains. We examine the mathematical properties of this model with varying experimental parameters. We simulate the growths of the yeast strains and compare them with the experimental results. We also compute and analyze the equilibrium state of the system and prove that it is asymptotically and evolutionarily stable.

  8. Virgin olive oil yeasts: A review.

    PubMed

    Ciafardini, Gino; Zullo, Biagi Angelo

    2018-04-01

    This review summarizes current knowledge on virgin olive oil yeasts. Newly produced olive oil contains solid particles and micro drops of vegetation water in which yeasts reproduce to become the typical microbiota of olive oil. To date, about seventeen yeast species have been isolated from different types of olive oils and their by-products, of which six species have been identified as new species. Certain yeast species contribute greatly to improving the sensorial characteristics of the newly produced olive oil, whereas other species are considered harmful as they can damage the oil quality through the production of unpleasant flavors and triacylglycerol hydrolysis. Studies carried out in certain yeast strains have demonstrated the presence of defects in olive oil treated with Candida adriatica, Nakazawaea wickerhamii and Candida diddensiae specific strains, while other olive oil samples treated with other Candida diddensiae strains were defect-free after four months of storage and categorized as extra virgin. A new acetic acid producing yeast species, namely, Brettanomyces acidodurans sp. nov., which was recently isolated from olive oil, could be implicated in the wine-vinegary defect of the product. Other aspects related to the activity of the lipase-producing yeasts and the survival of the yeast species in the flavored olive oils are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract is the food ingredient resulting from concentration of the solubles of mechanically ruptured cells of a selected strain of yeast, Saccharomyces...

  10. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b) The...

  11. 21 CFR 184.1983 - Bakers yeast extract.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bakers yeast extract. 184.1983 Section 184.1983... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract... a selected strain of yeast, Saccharomyces cerevisiae. It may be concentrated or dried. (b) The...

  12. History of genome editing in yeast.

    PubMed

    Fraczek, Marcin G; Naseeb, Samina; Delneri, Daniela

    2018-05-01

    For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30-40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non-conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high-throughput PCR-based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this 'Budding topic' we discuss the significant developments of genome editing in yeast, mainly focusing on Cre-loxP mediated recombination, delitto perfetto and CRISPR/Cas. © 2018 The Authors. Yeast published by John Wiley & Sons, Ltd.

  13. Inventions on baker's yeast strains and specialty ingredients.

    PubMed

    Gélinas, Pierre

    2009-06-01

    Baker's yeast is one of the oldest food microbial starters. Between 1927 and 2008, 165 inventions on more than 337 baker's yeast strains were patented. The first generation of patented yeast strains claimed improved biomass yield at the yeast plant, higher gassing power in dough or better survival to drying to prepare active dry baker's yeast. Especially between 1980 and 1995, a major interest was given to strains for multiple bakery applications such as dough with variable sugar content and stored at refrigeration (cold) or freezing temperatures. During the same period, genetically engineered yeast strains became very popular but did not find applications in the baking industry. Since year 2000, patented baker's yeast strains claimed aroma, anti-moulding or nutritive properties to better meet the needs of the baking industry. In addition to patents on yeast strains, 47 patents were issued on baker's yeast specialty ingredients for niche markets. This review shows that patents on baker's yeast with improved characteristics such as aromatic or nutritive properties have regularly been issued since the 1920's. Overall, it also confirms recent interest for a very wide range of tailored-made yeast-based ingredients for bakery applications.

  14. Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

    DOEpatents

    Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

    2012-03-20

    Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

  15. Experimental evolution of a sexually selected display in yeast

    PubMed Central

    Rogers, David W.; Greig, Duncan

    2008-01-01

    The fundamental principle underlying sexual selection theory is that an allele conferring an advantage in the competition for mates will spread through a population. Remarkably, this has never been demonstrated empirically. We have developed an experimental system using yeast for testing genetic models of sexual selection. Yeast signal to potential partners by producing an attractive pheromone; stronger signallers are preferred as mates. We tested the effect of high and low levels of sexual selection on the evolution of a gene determining the strength of this signal. Under high sexual selection, an allele encoding a stronger signal was able to invade a population of weak signallers, and we observed a corresponding increase in the amount of pheromone produced. By contrast, the strong signalling allele failed to invade under low sexual selection. Our results demonstrate, for the first time, the spread of a sexually selected allele through a population, confirming the central assumption of sexual selection theory. Our yeast system is a powerful tool for investigating the genetics of sexual selection. PMID:18842545

  16. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

  17. 21 CFR 172.898 - Bakers yeast glycan.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

  18. Stochastic simulations of a synthetic bacteria-yeast ecosystem

    PubMed Central

    2012-01-01

    Background The field of synthetic biology has greatly evolved and numerous functions can now be implemented by artificially engineered cells carrying the appropriate genetic information. However, in order for the cells to robustly perform complex or multiple tasks, co-operation between them may be necessary. Therefore, various synthetic biological systems whose functionality requires cell-cell communication are being designed. These systems, microbial consortia, are composed of engineered cells and exhibit a wide range of behaviors. These include yeast cells whose growth is dependent on one another, or bacteria that kill or rescue each other, synchronize, behave as predator-prey ecosystems or invade cancer cells. Results In this paper, we study a synthetic ecosystem comprising of bacteria and yeast that communicate with and benefit from each other using small diffusible molecules. We explore the behavior of this heterogeneous microbial consortium, composed of Saccharomyces cerevisiae and Escherichia coli cells, using stochastic modeling. The stochastic model captures the relevant intra-cellular and inter-cellular interactions taking place in and between the eukaryotic and prokaryotic cells. Integration of well-characterized molecular regulatory elements into these two microbes allows for communication through quorum sensing. A gene controlling growth in yeast is induced by bacteria via chemical signals and vice versa. Interesting dynamics that are common in natural ecosystems, such as obligatory and facultative mutualism, extinction, commensalism and predator-prey like dynamics are observed. We investigate and report on the conditions under which the two species can successfully communicate and rescue each other. Conclusions This study explores the various behaviors exhibited by the cohabitation of engineered yeast and bacterial cells. The way that the model is built allows for studying the dynamics of any system consisting of two species communicating with one

  19. The wine and beer yeast Dekkera bruxellensis

    PubMed Central

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-01-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:24932634

  20. The wine and beer yeast Dekkera bruxellensis.

    PubMed

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.

  1. 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.

  2. Biotechnology of non-Saccharomyces yeasts-the basidiomycetes.

    PubMed

    Johnson, Eric A

    2013-09-01

    Yeasts are the major producer of biotechnology products worldwide, exceeding production in capacity and economic revenues of other groups of industrial microorganisms. Yeasts have wide-ranging fundamental and industrial importance in scientific, food, medical, and agricultural disciplines (Fig. 1). Saccharomyces is the most important genus of yeast from fundamental and applied perspectives and has been expansively studied. Non-Saccharomyces yeasts (non-conventional yeasts) including members of the Ascomycetes and Basidiomycetes also have substantial current utility and potential applicability in biotechnology. In an earlier mini-review, "Biotechnology of non-Saccharomyces yeasts-the ascomycetes" (Johnson Appl Microb Biotechnol 97: 503-517, 2013), the extensive biotechnological utility and potential of ascomycetous yeasts are described. Ascomycetous yeasts are particularly important in food and ethanol formation, production of single-cell protein, feeds and fodder, heterologous production of proteins and enzymes, and as model and fundamental organisms for the delineation of genes and their function in mammalian and human metabolism and disease processes. In contrast, the roles of basidiomycetous yeasts in biotechnology have mainly been evaluated only in the past few decades and compared to the ascomycetous yeasts and currently have limited industrial utility. From a biotechnology perspective, the basidiomycetous yeasts are known mainly for the production of enzymes used in pharmaceutical and chemical synthesis, for production of certain classes of primary and secondary metabolites such as terpenoids and carotenoids, for aerobic catabolism of complex carbon sources, and for bioremediation of environmental pollutants and xenotoxicants. Notwithstanding, the basidiomycetous yeasts appear to have considerable potential in biotechnology owing to their catabolic utilities, formation of enzymes acting on recalcitrant substrates, and through the production of unique primary

  3. Comparison of the Vitek 2 Antifungal Susceptibility System with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest Techniques for In Vitro Detection of Antifungal Resistance in Yeast Isolates ▿ ‖

    PubMed Central

    Cuenca-Estrella, Manuel; Gomez-Lopez, Alicia; Alastruey-Izquierdo, Ana; Bernal-Martinez, Leticia; Cuesta, Isabel; Buitrago, Maria J.; Rodriguez-Tudela, Juan L.

    2010-01-01

    The commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% for Candida spp. and stood at 92% for Cryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P < 0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for all Candida spp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST. PMID:20220169

  4. 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.

  5. Influence of Zero-Shear on Yeast Development

    NASA Technical Reports Server (NTRS)

    McGinnis, Michael R.

    1997-01-01

    The objective of the research was to begin evaluating the effect of zero-shear on the development of the cell wall of Saccharomyces cerevisiae employing the High Aspect Rotating-Wall Vessel (HARV) NASA bioreactor. This particular yeast has enormous potential for research as a model eukaryotic system on the International Space Station, as well as the production of food stuffs' at the future lunar colony. Because the cell wall is the barrier between the cell and the environment, its form and function as influenced by microgravity is of great importance. Morphologic studies revealed that the circularity and total area of the individual yeast cells were essentially the same in both the control and test HARV's. The growth rates were also essentially the same. In zero-shear, the yeast grew in clumps consisting of rudimentary pseudohyphae in contrast to solitary budding cells in the control. Based upon mechanical and sonic shear applied to the yeast cells, those grown in zero-shear had stronger cell walls and septa. This suggests that there are structural differences, most likely related to the chitin skeleton of the cell wall. From this research further NASA support was obtained to continue the work. Investigations will deal with gene expression and ultrastructure. These will lead to a clearer assessment of the value of S. cerevisiae eukaryotic as a model for space station research.

  6. Continuous beer fermentation using immobilized yeast cell bioreactor systems.

    PubMed

    Brányik, Tomás; Vicente, António A; Dostálek, Pavel; Teixeira, José A

    2005-01-01

    Traditional beer fermentation and maturation processes use open fermentation and lager tanks. Although these vessels had previously been considered indispensable, during the past decades they were in many breweries replaced by large production units (cylindroconical tanks). These have proved to be successful, both providing operating advantages and ensuring the quality of the final beer. Another promising contemporary technology, namely, continuous beer fermentation using immobilized brewing yeast, by contrast, has found only a limited number of industrial applications. Continuous fermentation systems based on immobilized cell technology, albeit initially successful, were condemned to failure for several reasons. These include engineering problems (excess biomass and problems with CO(2) removal, optimization of operating conditions, clogging and channeling of the reactor), unbalanced beer flavor (altered cell physiology, cell aging), and unrealized cost advantages (carrier price, complex and unstable operation). However, recent development in reactor design and understanding of immobilized cell physiology, together with application of novel carrier materials, could provide a new stimulus to both research and application of this promising technology.

  7. Between science and industry-applied yeast research.

    PubMed

    Korhola, Matti

    2018-03-01

    I was fortunate to enter yeast research at the Alko Research Laboratories with a strong tradition in yeast biochemistry and physiology studies. At the same time in the 1980s there was a fundamental or paradigm change in molecular biology research with discoveries in DNA sequencing and other analytical and physical techniques for studying macromolecules and cells. Since that time biotechnological research has expanded the traditional fermentation industries to efficient production of industrial and other enzymes and specialty chemicals. Our efforts were directed towards improving the industrial production organisms: minerals enriched yeasts (Se, Cr, Zn) and high glutathione content yeast, baker´s, distiller´s, sour dough and wine yeasts, and the fungal Trichoderma reesei platform for enzyme production. I am grateful for the trust of my colleagues in several leadership positions at the Alko Research Laboratories, Yeast Industry Platform and at the international yeast community.

  8. Applying operations research to optimize a novel population management system for cancer screening

    PubMed Central

    Zai, Adrian H; Kim, Seokjin; Kamis, Arnold; Hung, Ken; Ronquillo, Jeremiah G; Chueh, Henry C; Atlas, Steven J

    2014-01-01

    Objective To optimize a new visit-independent, population-based cancer screening system (TopCare) by using operations research techniques to simulate changes in patient outreach staffing levels (delegates, navigators), modifications to user workflow within the information technology (IT) system, and changes in cancer screening recommendations. Materials and methods TopCare was modeled as a multiserver, multiphase queueing system. Simulation experiments implemented the queueing network model following a next-event time-advance mechanism, in which systematic adjustments were made to staffing levels, IT workflow settings, and cancer screening frequency in order to assess their impact on overdue screenings per patient. Results TopCare reduced the average number of overdue screenings per patient from 1.17 at inception to 0.86 during simulation to 0.23 at steady state. Increases in the workforce improved the effectiveness of TopCare. In particular, increasing the delegate or navigator staff level by one person improved screening completion rates by 1.3% or 12.2%, respectively. In contrast, changes in the amount of time a patient entry stays on delegate and navigator lists had little impact on overdue screenings. Finally, lengthening the screening interval increased efficiency within TopCare by decreasing overdue screenings at the patient level, resulting in a smaller number of overdue patients needing delegates for screening and a higher fraction of screenings completed by delegates. Conclusions Simulating the impact of changes in staffing, system parameters, and clinical inputs on the effectiveness and efficiency of care can inform the allocation of limited resources in population management. PMID:24043318

  9. Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

    PubMed

    Hopper, A K; Schultz, L D; Shapiro, R A

    1980-03-01

    By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

  10. Solving ethanol production problems with genetically modified yeast strains

    PubMed Central

    Abreu-Cavalheiro, A.; Monteiro, G.

    2013-01-01

    The current world demand for bioethanol is increasing as a consequence of low fossil fuel availability and a growing number of ethanol/gasoline flex-fuel cars. In addition, countries in several parts of the world have agreed to reduce carbon dioxide emissions, and the use of ethanol as a fuel (which produces fewer pollutants than petroleum products) has been considered to be a good alternative to petroleum products. The ethanol that is produced in Brazil from the first-generation process is optimized and can be accomplished at low cost. However, because of the large volume of ethanol that is produced and traded each year, any small improvement in the process could represent a savings of billions dollars. Several Brazilian research programs are investing in sugarcane improvement, but little attention has been given to the improvement of yeast strains that participate in the first-generation process at present. The Brazilian ethanol production process uses sugarcane as a carbon source for the yeast Saccharomyces cerevisiae. Yeast is then grown at a high cellular density and high temperatures in large-capacity open tanks with cells recycle. All of these culture conditions compel the yeast to cope with several types of stress. Among the main stressors are high temperatures and high ethanol concentrations inside the fermentation tanks during alcohol production. Moreover, the competition between the desired yeast strains, which are inoculated at the beginning of the process, with contaminants such as wild type yeasts and bacteria, requires acid treatment to successfully recycle the cells. This review is focused on describing the problems and stressors within the Brazilian ethanol production system. It also highlights some genetic modifications that can help to circumvent these difficulties in yeast. PMID:24516432

  11. Solving ethanol production problems with genetically modified yeast strains.

    PubMed

    Abreu-Cavalheiro, A; Monteiro, G

    2013-01-01

    The current world demand for bioethanol is increasing as a consequence of low fossil fuel availability and a growing number of ethanol/gasoline flex-fuel cars. In addition, countries in several parts of the world have agreed to reduce carbon dioxide emissions, and the use of ethanol as a fuel (which produces fewer pollutants than petroleum products) has been considered to be a good alternative to petroleum products. The ethanol that is produced in Brazil from the first-generation process is optimized and can be accomplished at low cost. However, because of the large volume of ethanol that is produced and traded each year, any small improvement in the process could represent a savings of billions dollars. Several Brazilian research programs are investing in sugarcane improvement, but little attention has been given to the improvement of yeast strains that participate in the first-generation process at present. The Brazilian ethanol production process uses sugarcane as a carbon source for the yeast Saccharomyces cerevisiae. Yeast is then grown at a high cellular density and high temperatures in large-capacity open tanks with cells recycle. All of these culture conditions compel the yeast to cope with several types of stress. Among the main stressors are high temperatures and high ethanol concentrations inside the fermentation tanks during alcohol production. Moreover, the competition between the desired yeast strains, which are inoculated at the beginning of the process, with contaminants such as wild type yeasts and bacteria, requires acid treatment to successfully recycle the cells. This review is focused on describing the problems and stressors within the Brazilian ethanol production system. It also highlights some genetic modifications that can help to circumvent these difficulties in yeast.

  12. Standard Printing Screen System.

    DTIC Science & Technology

    area pattern screens. It also describes the creation of a 100-step continuous growth halftone scale for the purpose of specifying quality control tolerances of screen tints for the printed product. (Author)

  13. The Red and White Yeast Lab: An Introduction to Science as a Process.

    ERIC Educational Resources Information Center

    White, Brian T.

    1999-01-01

    Describes an experimental system based on an engineered strain of bakers' yeast that is designed to involve students in the process by which scientific knowledge is generated. Students are asked to determine why the yeast grow to form a reproducible pattern of red and white. (WRM)

  14. 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

  15. Restructuring the Ikeda City school urinary screening system: report of a screening survey

    PubMed Central

    2013-01-01

    Background Annual urinary screening is conducted at municipal kindergartens, elementary schools, and junior high schools in Ikeda City, Osaka, Japan (Ikeda City School System), and the results are reviewed by a general physician, but standards for when to recommend specialist referral have not been clear. Methods In all children attending the Ikeda City School System in 2012, dipstick urinalysis of a first-morning urine specimen was recommended once or twice, and if a second urinalysis showed proteinuria (≥1+), the urinary protein/creatinine ratio was measured. If this showed ≥0.2 g/g of creatinine (g/gCr), it was recommended that the child be evaluated by a specialist at Ikeda City Hospital. Results Urinary screening was performed in about 20% (388) of kindergarten, about 90% (5363) of elementary school, and about 86% (2523) of junior high school children living in Ikeda City. Urine samples were obtained from 387, 5349, and 2476 children, respectively. The urinary protein/creatinine ratio was ≥0.2 g/gCr in 13 children, including 1 elementary and 12 junior high children. In these 13 children, chronic nephritic syndrome (CNS) was suspected in 6 junior high school children, and of these, this was a new finding in 5, and renal biopsy was indicated in 3. In Ikeda City, the prevalence of CNS in elementary school children was <0.03%, the prevalence of CNS in junior high school children was 0.29%, and a renal biopsy was indicated in 0.14%. By eliminating the costs associated with assessment of the results by the Ikeda Medical Association, and by directly contracting with the testing company, the expenses paid by Ikeda City for the system itself decreased from 2,508,619 yen to 966,157 yen. Conclusions Incorporating the urinary protein/creatinine ratio into the school urinary screening system in the Ikeda City School System and clarifying standards for specialist referral has enabled restructuring of the system so that is efficient and its effectiveness can be

  16. Yeast-based biosensors: design and applications.

    PubMed

    Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

    2015-02-01

    Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  17. Proteolysis suppresses spontaneous prion generation in yeast.

    PubMed

    Okamoto, Atsushi; Hosoda, Nao; Tanaka, Anri; Newnam, Gary P; Chernoff, Yury O; Hoshino, Shin-Ichi

    2017-12-08

    Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [ PSI + ] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [ PSI + ] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA ( pep4 Δ) or PrB ( prb1 Δ), increased the rate of de novo formation of [ PSI + ] prion up to ∼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [ PSI + ] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [ PSI + ] strains nor eliminated preexisting [ PSI + ]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [ PSI + ] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Quality improvement and geographical indication of cachaça (Brazilian spirit) by using locally selected yeast strains.

    PubMed

    Barbosa, E A; Souza, M T; Diniz, R H S; Godoy-Santos, F; Faria-Oliveira, F; Correa, L F M; Alvarez, F; Coutrim, M X; Afonso, R J C F; Castro, I M; Brandão, R L

    2016-10-01

    In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachaça production). Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachaça. Molecular polymorphic analyses were performed to characterize these isolates. Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachaça production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. The application of selected yeast to cachaça production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. This study suggests that the use of selected yeast strains could contribute to obtain a cachaça similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation. © 2016 The Society for Applied Microbiology.

  19. Microfluidic Devices for Drug Delivery Systems and Drug Screening

    PubMed Central

    Kompella, Uday B.; Damiati, Safa A.

    2018-01-01

    Microfluidic devices present unique advantages for the development of efficient drug carrier particles, cell-free protein synthesis systems, and rapid techniques for direct drug screening. Compared to bulk methods, by efficiently controlling the geometries of the fabricated chip and the flow rates of multiphase fluids, microfluidic technology enables the generation of highly stable, uniform, monodispersed particles with higher encapsulation efficiency. Since the existing preclinical models are inefficient drug screens for predicting clinical outcomes, microfluidic platforms might offer a more rapid and cost-effective alternative. Compared to 2D cell culture systems and in vivo animal models, microfluidic 3D platforms mimic the in vivo cell systems in a simple, inexpensive manner, which allows high throughput and multiplexed drug screening at the cell, organ, and whole-body levels. In this review, the generation of appropriate drug or gene carriers including different particle types using different configurations of microfluidic devices is highlighted. Additionally, this paper discusses the emergence of fabricated microfluidic cell-free protein synthesis systems for potential use at point of care as well as cell-, organ-, and human-on-a-chip models as smart, sensitive, and reproducible platforms, allowing the investigation of the effects of drugs under conditions imitating the biological system. PMID:29462948

  20. 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.

  1. Automated Age-related Macular Degeneration screening system using fundus images.

    PubMed

    Kunumpol, P; Umpaipant, W; Kanchanaranya, N; Charoenpong, T; Vongkittirux, S; Kupakanjana, T; Tantibundhit, C

    2017-07-01

    This work proposed an automated screening system for Age-related Macular Degeneration (AMD), and distinguishing between wet or dry types of AMD using fundus images to assist ophthalmologists in eye disease screening and management. The algorithm employs contrast-limited adaptive histogram equalization (CLAHE) in image enhancement. Subsequently, discrete wavelet transform (DWT) and locality sensitivity discrimination analysis (LSDA) were used to extract features for a neural network model to classify the results. The results showed that the proposed algorithm was able to distinguish between normal eyes, dry AMD, or wet AMD with 98.63% sensitivity, 99.15% specificity, and 98.94% accuracy, suggesting promising potential as a medical support system for faster eye disease screening at lower costs.

  2. Preparation of corncob grits as a carrier for immobilizing yeast cells for ethanol production.

    PubMed

    Lee, Sang-Eun; Lee, Choon Geun; Kang, Do Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

    2012-12-01

    In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

  3. [Continuous ethanol fermentation coupled with recycling of yeast flocs].

    PubMed

    Wang, Bo; Ge, Xu-Meng; Li, Ning; Bai, Feng-Wu

    2006-09-01

    A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220g/L of reducing sugar, supplemented with 1.5g/L (NH4)2HPO4 and 2.5g/L KH2PO4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057h(-1). The yeast flocs separated by sedimentation were recycled into the first fermentor as two different models: activation-recycle and direct recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at the high dilution rates. The broth containing more than 101g/L ethanol, 3.2g/L residual reducing sugar and 7.7g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77g/(L x h), which increased by more than 70% compared with that achieved in the same tank in series system without recycling of yeast cells.

  4. Oral yeast colonization throughout pregnancy.

    PubMed

    Rio, R; Simões-Silva, L; Garro, S; Silva, M-J; Azevedo, Á; Sampaio-Maia, B

    2017-03-01

    Recent studies suggest that placenta may harbour a unique microbiome that may have origin in maternal oral microbiome. Although the major physiological and hormonal adjustments observed in pregnant women lead to biochemical and microbiological modifications of the oral environment, very few studies evaluated the changes suffered by the oral microbiota throughout pregnancy. So, the aim of our study was to evaluate oral yeast colonization throughout pregnancy and to compare it with non-pregnant women. The oral yeast colonization was assessed in saliva of 30 pregnant and non-pregnant women longitudinally over a 6-months period. Demographic information was collected, a non-invasive intra-oral examination was performed and saliva flow and pH were determined. Pregnant and non-pregnant groups were similar regarding age and level of education. Saliva flow rate did not differ, but saliva pH was lower in pregnant than in non-pregnant women. Oral yeast prevalence was higher in pregnant than in non-pregnant women, either in the first or in the third trimester, but did not attain statistical significance. In individuals colonized with yeast, the total yeast quantification (Log10CFU/mL) increase from the 1st to the 3rd trimester in pregnant women, but not in non-pregnant women. Pregnancy may favour oral yeast growth that may be associated with an acidic oral environment.

  5. Biotechnological Applications of Dimorphic Yeasts

    NASA Astrophysics Data System (ADS)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  6. Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

    PubMed

    Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

    2014-03-20

    Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Yeasts of the soil – obscure but precious

    PubMed Central

    2018-01-01

    Abstract Pioneering studies performed in the nineteenth century demonstrated that yeasts are present in below‐ground sources. Soils were regarded more as a reservoir for yeasts that reside in habitats above it. Later studies showed that yeast communities in soils are taxonomically diverse and different from those above‐ground. Soil yeasts possess extraordinary adaptations that allow them to survive in a wide range of environmental conditions. A few species are promising sources of yeast oils and have been used in agriculture as potential antagonists of soil‐borne plant pathogens or as plant growth promoters. Yeasts have been studied mainly in managed soils such as vineyards, orchards and agricultural fields, and to a lesser extent under forests and grasslands. Our knowledge of soil yeasts is further biased towards temperate and boreal forests, whereas data from Africa, the Americas and Asia are scarce. Although soil yeast communities are often species‐poor in a single sample, they are more diverse on the biotope level. Soil yeasts display pronounced endemism along with a surprisingly high proportion of currently unidentified species. However, like other soil inhabitants, yeasts are threatened by habitat alterations owing to anthropogenic activities such as agriculture, deforestation and urbanization. In view of the rapid decline of many natural habitats, the study of soil yeasts in undisturbed or low‐managed biotopes is extremely valuable. The purpose of this review is to encourage researchers, both biologists and soil scientists, to include soil yeasts in future studies. PMID:29365211

  8. Yeast Toxicogenomics: Genome-Wide Responses to Chemical Stresses with Impact in Environmental Health, Pharmacology, and Biotechnology

    PubMed Central

    dos Santos, Sandra C.; Teixeira, Miguel Cacho; Cabrito, Tânia R.; Sá-Correia, Isabel

    2012-01-01

    The emerging transdisciplinary field of Toxicogenomics aims to study the cell response to a given toxicant at the genome, transcriptome, proteome, and metabolome levels. This approach is expected to provide earlier and more sensitive biomarkers of toxicological responses and help in the delineation of regulatory risk assessment. The use of model organisms to gather such genomic information, through the exploitation of Omics and Bioinformatics approaches and tools, together with more focused molecular and cellular biology studies are rapidly increasing our understanding and providing an integrative view on how cells interact with their environment. The use of the model eukaryote Saccharomyces cerevisiae in the field of Toxicogenomics is discussed in this review. Despite the limitations intrinsic to the use of such a simple single cell experimental model, S. cerevisiae appears to be very useful as a first screening tool, limiting the use of animal models. Moreover, it is also one of the most interesting systems to obtain a truly global understanding of the toxicological response and resistance mechanisms, being in the frontline of systems biology research and developments. The impact of the knowledge gathered in the yeast model, through the use of Toxicogenomics approaches, is highlighted here by its use in prediction of toxicological outcomes of exposure to pesticides and pharmaceutical drugs, but also by its impact in biotechnology, namely in the development of more robust crops and in the improvement of yeast strains as cell factories. PMID:22529852

  9. A cloud-based system for automatic glaucoma screening.

    PubMed

    Fengshou Yin; Damon Wing Kee Wong; Ying Quan; Ai Ping Yow; Ngan Meng Tan; Gopalakrishnan, Kavitha; Beng Hai Lee; Yanwu Xu; Zhuo Zhang; Jun Cheng; Jiang Liu

    2015-08-01

    In recent years, there has been increasing interest in the use of automatic computer-based systems for the detection of eye diseases including glaucoma. However, these systems are usually standalone software with basic functions only, limiting their usage in a large scale. In this paper, we introduce an online cloud-based system for automatic glaucoma screening through the use of medical image-based pattern classification technologies. It is designed in a hybrid cloud pattern to offer both accessibility and enhanced security. Raw data including patient's medical condition and fundus image, and resultant medical reports are collected and distributed through the public cloud tier. In the private cloud tier, automatic analysis and assessment of colour retinal fundus images are performed. The ubiquitous anywhere access nature of the system through the cloud platform facilitates a more efficient and cost-effective means of glaucoma screening, allowing the disease to be detected earlier and enabling early intervention for more efficient intervention and disease management.

  10. 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.

  11. Yeasts in floral nectar: a quantitative survey

    PubMed Central

    Herrera, Carlos M.; de Vega, Clara; Canto, Azucena; Pozo, María I.

    2009-01-01

    Background and Aims One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. Methods The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. Key Results Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 105 cells mm−3). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 104 cells mm−3 were commonplace, and densities >105 cells mm−3 were not rare. About one-fifth of species at each site had mean yeast cell densities >104 cells mm−3. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. Conclusions Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence

  12. 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...

  13. Use of the BioMerieux ID 32C yeast identification system for identification of aerobic actinomycetes of medical importance.

    PubMed Central

    Muir, D B; Pritchard, R C

    1997-01-01

    The BioMerieux ID 32C Yeast Identification System was examined to determine its usefulness as a rapid method for the identification of medically important aerobic actinomycetes. More than 290 strains were tested by this method and the results were compared to those obtained by conventional methods. It was found that aerobic actinomycetes could be differentiated to species level in 7 days by the ID 32C system. PMID:9399526

  14. 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.

  15. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

    PubMed

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  16. 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®.

  17. 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

  18. 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.

  19. Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges

    PubMed Central

    Montanez-Sauri, Sara I.; Beebe, David J.; Sung, Kyung Eun

    2015-01-01

    The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g. spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments. PMID:25274061

  20. Yeast mitochondria: an overview of mitochondrial biology and the potential of mitochondrial systems biology.

    PubMed

    Malina, Carl; Larsson, Christer; Nielsen, Jens

    2018-08-01

    Mitochondria are dynamic organelles of endosymbiotic origin that are essential components of eukaryal cells. They contain their own genetic machinery, have multicopy genomes and like their bacterial ancestors they consist of two membranes. However, the majority of the ancestral genome has been lost or transferred to the nuclear genome of the host, preserving only a core set of genes involved in oxidative phosphorylation. Mitochondria perform numerous biological tasks ranging from bioenergetics to production of protein co-factors, including heme and iron-sulfur clusters. Due to the importance of mitochondria in many cellular processes, mitochondrial dysfunction is implicated in a wide variety of human disorders. Much of our current knowledge on mitochondrial function and dysfunction comes from studies using Saccharomyces cerevisiae. This yeast has good fermenting capacity, rendering tolerance to mutations that inactivate oxidative phosphorylation and complete loss of mitochondrial DNA. Here, we review yeast mitochondrial metabolism and function with focus on S. cerevisiae and its contribution in understanding mitochondrial biology. We further review how systems biology studies, including mathematical modeling, has allowed gaining new insight into mitochondrial function, and argue that this approach may enable us to gain a holistic view on how mitochondrial function interacts with different cellular processes.

  1. Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

    PubMed Central

    Chen, Chen; Lim, Hong Hwa; Shi, Jian; Tamura, Sachiko; Maeshima, Kazuhiro; Surana, Uttam; Gan, Lu

    2016-01-01

    Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin—nucleosomes—are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an “open” configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome–nucleosome associations. PMID:27605704

  2. Study of an overload truck screening system for a sustainable highway bridge network.

    DOT National Transportation Integrated Search

    2009-01-01

    he objective of this project was to study the use and implementation of an overload truck screening system for bridge structures. This screening system combines two advanced technologies, namely a weigh-in-motion (WIM) system and a bridge strain sens...

  3. 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.

  4. Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

    PubMed

    Hilber-Bodmer, Maja; Schmid, Michael; Ahrens, Christian H; Freimoser, Florian M

    2017-01-05

    While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes. Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source. The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from

  5. Unraveling the Enzymatic Basis of Wine “Flavorome”: A Phylo-Functional Study of Wine Related Yeast Species

    PubMed Central

    Belda, Ignacio; Ruiz, Javier; Alastruey-Izquierdo, Ana; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-01-01

    Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the β-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, β-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species

  6. Correlating yeast cell stress physiology to changes in the cell surface morphology: atomic force microscopic studies.

    PubMed

    Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

    2006-07-06

    Atomic Force Microscopy (AFM) has emerged as a powerful biophysical tool in biotechnology and medicine to investigate the morphological, physical, and mechanical properties of yeasts and other biological systems. However, properties such as, yeasts' response to environmental stresses, metabolic activities of pathogenic yeasts, cell-cell/cell-substrate adhesion, and cell-flocculation have rarely been investigated so far by using biophysical tools. Our recent results obtained by AFM on one strain each of Saccharomyces cerevisiae and Schizosaccharomyces pombe show a clear correlation between the physiology of environmentally stressed yeasts and the changes in their surface morphology. The future directions of the AFM related techniques in relation to yeasts are also discussed.

  7. [Mitochondria inheritance in yeast saccharomyces cerevisiae].

    PubMed

    Fizikova, A Iu

    2011-01-01

    The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.

  8. Building Better Biosensors for Exploration into Deep-Space, Using Humanized Yeast

    NASA Technical Reports Server (NTRS)

    Liddell, Lauren; Santa Maria, Sergio; Tieze, Sofia; Bhattacharya, Sharmila

    2017-01-01

    1.BioSentinel is 1 of 13 secondary payloads hitching a ride beyond Low Earth Orbit on Exploration Mission 1 (EM-1), set to launch from NASAs Space Launch System in 2019. EM-1 is our first opportunity to investigate the effects of the deep space environment on a eukaryotic biological system, the budding yeast S. cerevisiae. Though separated by a billion years of evolution we share hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast is an ideal biosensor for detecting typesextent of damage induced by deep-space radiation.We will fly desiccated cells, then rehydrate to wake them up when the automated payload is ready to initiate the experiment. Rehydration solution contains SC (Synthetic Complete) media and alamarBlue, an indicator for changes in growth and metabolism. Telemetry of LED readings will then allow us to detect how cells respond throughout the mission. The desiccation-rehydration process can be extremely damaging to cells, and can severely diminish our ability to accurately measure and model cellular responses to deep-space radiation. The aim of this study is to develop a better biosensor: yeast strains that are more resistant to desiccation stress. We will over-express known cellular protectants, including hydrophilin Sip18, the protein disaggregase Hsp104, and thioredoxin Trx2, a responder to oxidative stress, then measure cell viability after desiccation to determine which factors improve stress tolerance. Over-expression of SIP18 in wine yeast starter cultures was previously reported to increase viability following desiccation stress by up to 70. Thus, we expect similar improvements in our space-yeast strains. By designing better yeast biosensors we can better prepare for and mitigate the potential dangers of deep-space radiation for future missions.This work is funded by NASAs AES program.

  9. Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

    PubMed

    Parry, J M; Sharp, D; Tippins, R S; Parry, E M

    1979-06-01

    A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.

  10. [Groups and sources of yeasts in house dust].

    PubMed

    Glushakova, A M; Zheltikova, T M; Chernov, I Iu

    2004-01-01

    House dust contains bacteria, mycelial fungi, microarthropods, and yeasts. The house dust samples collected in 25 apartments in Moscow and the Moscow region were found to contain yeasts belonging to the genera Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sporobolomyces, and Trichosporon. The most frequently encountered microorganisms were typical epiphytic yeasts, such as Cryptococcus diffluens and Rhodotorula mucilaginosa, which are capable of long-term preservation in an inactive state. The direct source of epiphytic yeasts occurring in the house dust might be the indoor plants, which were contaminated with these yeasts, albeit to a lesser degree than outdoor plants. Along with the typical epiphytic yeasts, the house dust contained the opportunistic yeast pathogens Candida catenulata, C. guillermondii, C. haemulonii, C. rugosa, and C. tropicalis, which are known as the causal agents of candidiasis. We failed to reveal any correlation between the abundance of particular yeast species in the house dust, residential characteristics, and the atopic dermatitis of the inhabitants.

  11. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    PubMed Central

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

    2015-01-01

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  12. Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs.

    PubMed

    White, L A; Newman, M C; Cromwell, G L; Lindemann, M D

    2002-10-01

    Brewers dried yeast, a source of mannan oligosaccharides (MOS), was assessed as an alternative to an antimicrobial agent (carbadox) for young pigs in two experiments. The yeast contained 5.2% MOS. Agglutination tests confirmed adsorption of several serovars of E. coli and Salmonella spp. onto the yeast product. In Exp. 1, seven replicates (five pigs per pen) of 22-d-old pigs were fed a nonmedicated basal diet or the basal diet with carbadox (55 mg/kg), yeast (3%), or a combination of 3% yeast and 2% citric acid for 28 d. Carbadox did not improve growth performance. Growth rate and feed intake were depressed (P < 0.05) in pigs fed yeast alone or in combination with acid. Log counts of total coliforms, Escherichia coli, and Clostridium perfringens in feces were not affected by diet, but Bifidobacteria spp. counts were lower (P < 0.05) in pigs fed the yeast + acid diet and lactobacilli counts were higher (P < 0.05) in pigs fed yeast. Fecal pH and VFA concentrations and intestinal morphological traits were not consistently affected by diet. Serum IgG levels were elevated in the yeast + acid (P < 0.01) group. In Exp. 2, the effects of yeast and carbadox additions to the diet on enteric microbial populations in young pigs housed in isolation units were evaluated. Pigs (n = 24) were weaned at 11 d of age (4.1 kg BW) and placed in isolation chambers (two pigs per chamber) equipped with individual air filtering systems and excrement containers. Treatments were a nonmedicated basal diet and the basal diet with 55 mg/kg of carbadox or with 3% yeast. Diets were fed for 29 d, then each pig was orally dosed with approximately 9.5 x 10(8) CFU of E. coli K88. Daily fecal E. coli K88 counts were not different (P > 0.05) among treatments, but fecal shedding of carbadox-resistant coliforms was higher (P < 0.01) during the 9-d period in pigs fed carbadox. Total fecal coliforms were consistently lower throughout the postinoculation period in pigs fed yeast (P < 0.05). Yeast reduced

  13. Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

    PubMed Central

    Melhem, MSC; Bertoletti, A; Lucca, HRL; Silva, RBO; Meneghin, FA; Szeszs, MW

    2013-01-01

    Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods. PMID:24688520

  14. Network-based reading system for lung cancer screening CT

    NASA Astrophysics Data System (ADS)

    Fujino, Yuichi; Fujimura, Kaori; Nomura, Shin-ichiro; Kawashima, Harumi; Tsuchikawa, Megumu; Matsumoto, Toru; Nagao, Kei-ichi; Uruma, Takahiro; Yamamoto, Shinji; Takizawa, Hotaka; Kuroda, Chikazumi; Nakayama, Tomio

    2006-03-01

    This research aims to support chest computed tomography (CT) medical checkups to decrease the death rate by lung cancer. We have developed a remote cooperative reading system for lung cancer screening over the Internet, a secure transmission function, and a cooperative reading environment. It is called the Network-based Reading System. A telemedicine system involves many issues, such as network costs and data security if we use it over the Internet, which is an open network. In Japan, broadband access is widespread and its cost is the lowest in the world. We developed our system considering human machine interface and security. It consists of data entry terminals, a database server, a computer aided diagnosis (CAD) system, and some reading terminals. It uses a secure Digital Imaging and Communication in Medicine (DICOM) encrypting method and Public Key Infrastructure (PKI) based secure DICOM image data distribution. We carried out an experimental trial over the Japan Gigabit Network (JGN), which is the testbed for the Japanese next-generation network, and conducted verification experiments of secure screening image distribution, some kinds of data addition, and remote cooperative reading. We found that network bandwidth of about 1.5 Mbps enabled distribution of screening images and cooperative reading and that the encryption and image distribution methods we proposed were applicable to the encryption and distribution of general DICOM images via the Internet.

  15. Network-assisted target identification for haploinsufficiency and homozygous profiling screens

    PubMed Central

    Wang, Sheng

    2017-01-01

    Chemical genomic screens have recently emerged as a systematic approach to drug discovery on a genome-wide scale. Drug target identification and elucidation of the mechanism of action (MoA) of hits from these noisy high-throughput screens remain difficult. Here, we present GIT (Genetic Interaction Network-Assisted Target Identification), a network analysis method for drug target identification in haploinsufficiency profiling (HIP) and homozygous profiling (HOP) screens. With the drug-induced phenotypic fitness defect of the deletion of a gene, GIT also incorporates the fitness defects of the gene’s neighbors in the genetic interaction network. On three genome-scale yeast chemical genomic screens, GIT substantially outperforms previous scoring methods on target identification on HIP and HOP assays, respectively. Finally, we showed that by combining HIP and HOP assays, GIT further boosts target identification and reveals potential drug’s mechanism of action. PMID:28574983

  16. Yeast 5 – an expanded reconstruction of the Saccharomyces cerevisiae metabolic network

    PubMed Central

    2012-01-01

    stoichiometrically constrained yeast metabolic model. It differs from previous reconstructions and models by emphasizing the distinction between the yeast metabolic reconstruction and the stoichiometrically constrained model, and makes both available as Additional file 4 and Additional file 5 and at http://yeast.sf.net/ as separate systems biology markup language (SBML) files. Through this separation, we intend to make the modeling process more accessible, explicit, transparent, and reproducible. PMID:22663945

  17. A search for H/ACA snoRNAs in yeast using MFE secondary structure prediction.

    PubMed

    Edvardsson, Sverker; Gardner, Paul P; Poole, Anthony M; Hendy, Michael D; Penny, David; Moulton, Vincent

    2003-05-01

    Noncoding RNA genes produce functional RNA molecules rather than coding for proteins. One such family is the H/ACA snoRNAs. Unlike the related C/D snoRNAs these have resisted automated detection to date. We develop an algorithm to screen the yeast genome for novel H/ACA snoRNAs. To achieve this, we introduce some new methods for facilitating the search for noncoding RNAs in genomic sequences which are based on properties of predicted minimum free-energy (MFE) secondary structures. The algorithm has been implemented and can be generalized to enable screening of other eukaryote genomes. We find that use of primary sequence alone is insufficient for identifying novel H/ACA snoRNAs. Only the use of secondary structure filters reduces the number of candidates to a manageable size. From genomic context, we identify three strong H/ACA snoRNA candidates. These together with a further 47 candidates obtained by our analysis are being experimentally screened.

  18. 21 CFR 172.590 - Yeast-malt sprout extract.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Yeast-malt sprout extract. 172.590 Section 172.590... Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this section, may... produced by partial hydrolysis of yeast extract (derived from Saccharomyces cereviseae, Saccharomyces...

  19. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    PubMed

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  20. Endosomal protein sorting and autophagy genes contribute to the regulation of yeast life span.

    PubMed

    Longo, Valter D; Nislow, Corey; Fabrizio, Paola

    2010-11-01

    Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.