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Sample records for cerevisiae prm1 homolog

  1. The Saccharomyces cerevisiae PRM1 homolog in Neurospora crassa is involved in vegetative and sexual cell fusion events but also has postfertilization functions.

    PubMed

    Fleissner, André; Diamond, Spencer; Glass, N Louise

    2009-02-01

    Cell-cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Delta mutants show an approximately 50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an approximately 50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with DeltaPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The DeltaPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a DeltaPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.

  2. Viable offspring obtained from Prm1-deficient sperm in mice

    PubMed Central

    Takeda, Naoki; Yoshinaga, Kazuya; Furushima, Kenryo; Takamune, Kazufumi; Li, Zhenghua; Abe, Shin-ichi; Aizawa, Shin-ichi; Yamamura, Ken-ichi

    2016-01-01

    Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1+/− male mice. Healthy Prm1+/− offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1+/− mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles. PMID:27250771

  3. The Yeast Cell Fusion Protein Prm1p Requires Covalent Dimerization to Promote Membrane Fusion

    PubMed Central

    Engel, Alex; Aguilar, Pablo S.; Walter, Peter

    2010-01-01

    Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion. PMID:20485669

  4. The role of centromere alignment in meiosis I segregation of homologous chromosomes in Saccharomyces cerevisiae.

    PubMed Central

    Guerra, C E; Kaback, D B

    1999-01-01

    During meiosis, homologous chromosomes pair and then segregate from each other at the first meiotic division. Homologous centromeres appear to be aligned when chromosomes are paired. The role of centromere alignment in meiotic chromosome segregation was investigated in Saccharomyces cerevisiae diploids that contained one intact copy of chromosome I and one copy bisected into two functional centromere-containing fragments. The centromere on one fragment was aligned with the centromere on the intact chromosome while the centromere on the other fragment was either aligned or misaligned. Fragments containing aligned centromeres segregated efficiently from the intact chromosome, while fragments containing misaligned centromeres segregated much less efficiently from the intact chromosome. Less efficient segregation was correlated with crossing over in the region between the misaligned centromeres. Models that suggest that these crossovers impede proper segregation by preventing either a segregation-promoting chromosome alignment on the meiotic spindle or some physical interaction between homologous centromeres are proposed. PMID:10581265

  5. Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2015-01-01

    The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples. PMID:26523287

  6. The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B2) uptake in Saccharomyces cerevisiae.

    PubMed

    Reihl, Petra; Stolz, Jürgen

    2005-12-01

    Riboflavin is a water-soluble vitamin (vitamin B2) required for the production of the flavin cofactors FMN and FAD. Mammals are unable to synthesize riboflavin and need a dietary supply of the vitamin. Riboflavin transport proteins operating in the plasma membrane thus have an important role in the absorption of the vitamin. However, their sequences remained elusive, and not a single eukaryotic riboflavin transporter is known to date. Here we used a genetic approach to isolate MCH5, a Saccharomyces cerevisiae gene with homology to mammalian monocarboxylate transporters, and characterize the protein as a plasma membrane transporter for riboflavin. This conclusion is based on the suppression of riboflavin biosynthetic mutants (rib mutants) by overexpression of MCH5 and by synthetic growth defects caused by deletion of MCH5 in rib mutants. We also show that cellular processes in multiple compartments are affected by deletion of MCH5 and localize the protein to the plasma membrane. Transport experiments in S. cerevisiae and Schizosaccharomyces pombe cells demonstrate that Mch5p is a high affinity transporter (Km = 17 microM) with a pH optimum at pH 7.5. Riboflavin uptake is not inhibited by protonophores, does not require metabolic energy, and operates by a facilitated diffusion mechanism. The expression of MCH5 is regulated by the cellular riboflavin content. This indicates that S. cerevisiae has a mechanism to sense riboflavin and avert riboflavin deficiency by increasing the expression of the plasma membrane transporter MCH5. Moreover, the other members of the MCH gene family appear to have unrelated functions.

  7. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

    PubMed Central

    Milne, G T; Jin, S; Shannon, K B; Weaver, D T

    1996-01-01

    DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells. PMID:8754818

  8. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6.

    PubMed Central

    van Gool, A J; Verhage, R; Swagemakers, S M; van de Putte, P; Brouwer, J; Troelstra, C; Bootsma, D; Hoeijmakers, J H

    1994-01-01

    Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity. Images PMID:7957102

  9. The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin.

    PubMed Central

    MacKay, V L; Welch, S K; Insley, M Y; Manney, T R; Holly, J; Saari, G C; Parker, M L

    1988-01-01

    Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of alpha factor, the peptide mating pheromone produced by mating-type alpha cells. We report here the DNA sequence of BAR1, the structural gene for barrier activity. The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases. Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site. Therefore, barrier protein is probably a protease that cleaves alpha factor. The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases. However, the BAR1 gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity. Images PMID:3124102

  10. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6.

    PubMed

    van Gool, A J; Verhage, R; Swagemakers, S M; van de Putte, P; Brouwer, J; Troelstra, C; Bootsma, D; Hoeijmakers, J H

    1994-11-15

    Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity. PMID:7957102

  11. The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

    PubMed

    Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

    2006-11-10

    Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

  12. Rad51 Inhibits Translocation Formation by Non-Conservative Homologous Recombination in Saccharomyces cerevisiae

    PubMed Central

    Manthey, Glenn M.; Bailis, Adam M.

    2010-01-01

    Chromosomal translocations are a primary biological response to ionizing radiation (IR) exposure, and are likely to result from the inappropriate repair of the DNA double-strand breaks (DSBs) that are created. An abundance of repetitive sequences in eukaryotic genomes provides ample opportunity for such breaks to be repaired by homologous recombination (HR) between non-allelic repeats. Interestingly, in the budding yeast, Saccharomyces cerevisiae the central strand exchange protein, Rad51 that is required for DSB repair by gene conversion between unlinked repeats that conserves genomic structure also suppresses translocation formation by several HR mechanisms. In particular, Rad51 suppresses translocation formation by single-strand annealing (SSA), perhaps the most efficient mechanism for translocation formation by HR in both yeast and mammalian cells. Further, the enhanced translocation formation that emerges in the absence of Rad51 displays a distinct pattern of genetic control, suggesting that this occurs by a separate mechanism. Since hypomorphic mutations in RAD51 in mammalian cells also reduce DSB repair by conservative gene conversion and stimulate non-conservative repair by SSA, this mechanism may also operate in humans and, perhaps contribute to the genome instability that propels the development of cancer. PMID:20686691

  13. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    PubMed

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

    2015-05-15

    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  14. Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein.

    PubMed Central

    Lipps, G; Krauss, G

    1999-01-01

    Adenylosuccinate synthase (EC 6.3.4.4) catalyses the first committed step in the synthesis of adenosine. We have overexpressed the cloned gene of Saccharomyces cerevisiae (ADE12) in S. cerevisiae. The recombinant enzyme exhibits similar kinetic behaviour to that of the native enzyme purified from S. cerevisiae. This ter-reactant dimeric enzyme shows Michaelis-Menten kinetics only with IMP. l-Aspartate and GTP display a weak negative co-operativity (Hill coefficient 0. 8-0.9). This negative co-operativity has not yet been reported for adenylosuccinate synthases from other organisms. Another unusual feature of the enzyme from S. cerevisiae is its negligible inhibition by adenine nucleotides and its pronounced inhibition by Cl(-) ions. PMID:10417315

  15. Isolation and Characterization of Two Saccharomyces Cerevisiae Genes Encoding Homologs of the Bacterial Hexa and Muts Mismatch Repair Proteins

    PubMed Central

    Reenan, R. A.; Kolodner, R. D.

    1992-01-01

    Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair. PMID:1459447

  16. Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

    PubMed Central

    Iuso, Domenico; Czernik, Marta; Toschi, Paola; Fidanza, Antonella; Zacchini, Federica; Feil, Robert; Curtet, Sandrine; Buchou, Thierry; Shiota, Hitoshi; Khochbin, Saadi; Ptak, Grazyna Ewa; Loi, Pasqualino

    2015-01-01

    Summary Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer. PMID:26628361

  17. Dynamics of homology searching during gene conversion in Saccharomyces cerevisiae revealed by donor competition.

    PubMed

    Coïc, Eric; Martin, Joshua; Ryu, Taehyun; Tay, Sue Yen; Kondev, Jané; Haber, James E

    2011-12-01

    One of the least understood aspects of homologous recombination is the process by which the ends of a double-strand break (DSB) search the entire genome for homologous templates that can be used to repair the break. We took advantage of the natural competition between the alternative donors HML and HMR employed during HO endonuclease-induced switching of the budding yeast MAT locus. The strong mating-type-dependent bias in the choice of the donors is enforced by the recombination enhancer (RE), which lies 17 kb proximal to HML. We investigated factors that improve the use of the disfavored donor. We show that the normal heterochromatic state of the donors does not impair donor usage, as donor choice is not affected by removing this epigenetic silencing. In contrast, increasing the length of homology shared by the disfavored donor increases its use. This result shows that donor choice is not irrevocable and implies that there are several encounters between the DSB ends and even the favored donor before recombination is accomplished. The increase by adding more homology is not linear; these results can be explained by a thermodynamic model that determines the energy cost of using one donor over the other. An important inference from this analysis is that when HML is favored as the donor, RE causes a reduction in its effective genomic distance from MAT from 200 kb to ∼20 kb, which we hypothesize occurs after the DSB is created, by epigenetic chromatin modifications around MAT.

  18. The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

    PubMed Central

    Carr, A M; Schmidt, H; Kirchhoff, S; Muriel, W J; Sheldrick, K S; Griffiths, D J; Basmacioglu, C N; Subramani, S; Clegg, M; Nasim, A

    1994-01-01

    The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity. Images PMID:8114734

  19. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination*

    PubMed Central

    Zhang, Qiang; Chen, Qi-he; Fu, Ming-liang; Wang, Jin-ling; Zhang, Hong-bo; He, Guo-qing

    2008-01-01

    The bglS gene encoding endo-l,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1S), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-l,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer. PMID:18600782

  20. An Inhibited Conformation for the Protein Kinase Domain of the Saccharomyces cerevisiae AMPK Homolog Snf1

    SciTech Connect

    Rudolph, M.; Amodeo, G; Tong, L

    2010-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic ({alpha}) subunit of AMPK/SNF1 in yeast (Snf1) contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID) and a region that mediates interactions with the two regulatory ({beta} and {gamma}) subunits. Here, the crystal structure of residues 41-440 of Snf1, which include the KD and AID, is reported at 2.4 {angstrom} resolution. The AID is completely disordered in the crystal. A new inhibited conformation of the KD is observed in a DFG-out conformation and with the glycine-rich loop adopting a structure that blocks ATP binding to the active site.

  1. Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5

    PubMed Central

    Jeong, Jee-Yeong; Johns, Jeremiah; Sinclair, Christopher; Park, Jung-Min; Rossie, Sandra

    2003-01-01

    Background Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level. Results The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth. Conclusions Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests

  2. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    PubMed

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

    2013-12-01

    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

  3. Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

    PubMed Central

    Mio, T; Adachi-Shimizu, M; Tachibana, Y; Tabuchi, H; Inoue, S B; Yabe, T; Yamada-Okabe, T; Arisawa, M; Watanabe, T; Yamada-Okabe, H

    1997-01-01

    Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase. PMID:9209021

  4. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae

    PubMed Central

    2016-01-01

    Most human somatic cells express insufficient levels of telomerase, which can result in telomere shortening and eventually senescence, both of which are hallmarks of ageing. Homology-directed repair (HDR) is important for maintaining proper telomere function in yeast and mammals. In Saccharomyces cerevisiae, Rad52 is required for almost all HDR mechanisms, and telomerase-null cells senesce faster in the absence of Rad52. However, its role in preventing accelerated senescence has been unclear. In this study, we make use of rad52 separation-of-function mutants to find that multiple Rad52-mediated HDR mechanisms are required to delay senescence, including break-induced replication and sister chromatid recombination. In addition, we show that misregulation of histone 3 lysine 56 acetylation, which is known to be defective in sister chromatid recombination, also causes accelerated senescence. We propose a model where Rad52 is needed to repair telomere attrition-induced replication stress. PMID:27428329

  5. pdc1(0) mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1.

    PubMed Central

    Seeboth, P G; Bohnsack, K; Hollenberg, C P

    1990-01-01

    The PDC1 gene coding for a pyruvate decarboxylase (PDC; EC 4.1.1.1) was deleted from the Saccharomyces cerevisiae genome. The resulting pdc1(0) mutants were able to grow on glucose and still contained 60 to 70% of the wild-type PDC activity. Two DNA fragments with sequences homologous to that of the PDC1 gene were cloned from the yeast genome. One of the cloned genes (PDC5) was expressed at high rates predominantly in pdc1(0) strains and probably encodes the remaining PDC activity in these strains. Expression from the PDC1 promoter in PDC1 wild-type and pdc1(0) strains was examined by the use of two reporter genes. Deletion of PDC1 led to increased expression of the two reporter genes regardless of whether the fusions were integrated into the genome or present on autonomously replicating plasmids. The results suggested that this effect was due to feedback regulation of the PDC1 promoter-driven expression in S. cerevisiae pdc1(0) strains. The yeast PDC1 gene was expressed in Escherichia coli, leading to an active PDC. This result shows that the PDC1-encoded subunit alone can form an active tetramer without yeast-specific processing steps. Images FIG. 1 FIG. 4 FIG. 5 FIG. 6 PMID:2404950

  6. Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins.

    PubMed

    Chooi, W Y; Otaka, E

    1984-11-01

    Specific antibodies directed against Drosophila melanogaster acidic ribosomal protein S14 were used in a comparative study of eucaryotic and procaryotic ribosomes by immunoblotting and enzyme-linked immunosorbent assays. Common antigenic determinants and, thus, structural homology were found between D. melanogaster, Saccharomyces cerevisiae (S25), rabbit liver (S12), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomes.

  7. MrpL36p, a highly diverged L31 ribosomal protein homolog with additional functional domains in Saccharomyces cerevisiae mitochondria.

    PubMed Central

    Williams, Elizabeth H; Perez-Martinez, Xochitl; Fox, Thomas D

    2004-01-01

    Translation in mitochondria utilizes a large complement of ribosomal proteins. Many mitochondrial ribosomal components are clearly homologous to eubacterial ribosomal proteins, but others appear unique to the mitochondrial system. A handful of mitochondrial ribosomal proteins appear to be eubacterial in origin but to have evolved additional functional domains. MrpL36p is an essential mitochondrial ribosomal large-subunit component in Saccharomyces cerevisiae. Increased dosage of MRPL36 also has been shown to suppress certain types of translation defects encoded within the mitochondrial COX2 mRNA. A central domain of MrpL36p that is similar to eubacterial ribosomal large-subunit protein L31 is sufficient for general mitochondrial translation but not suppression, and proteins bearing this domain sediment with the ribosomal large subunit in sucrose gradients. In contrast, proteins lacking the L31 domain, but retaining a novel N-terminal sequence and a C-terminal sequence with weak similarity to the Escherichia coli signal recognition particle component Ffh, are sufficient for dosage suppression and do not sediment with the large subunit of the ribosome. Interestingly, the activity of MrpL36p as a dosage suppressor exhibits gene and allele specificity. We propose that MrpL36p represents a highly diverged L31 homolog with derived domains functioning in mRNA selection in yeast mitochondria. PMID:15166137

  8. Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog.

    PubMed

    Abe, Yoichiro; Kita, Yoshiko; Niikura, Takako

    2008-01-01

    Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent protein that was fused to each C terminus of these proteins. Gup1 and Skn were co-localized with an endoplasmic reticulum marker, 78 kDa glucose-regulated protein, suggesting that these two molecules interact with overlapped targets, including Shh. In fact, full-length Shh coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using anti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N-terminal signaling domain of Shh under denaturing conditions. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results suggest that Gup1 is a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh.

  9. Isolation of the Candida albicans homologs of Saccharomyces cerevisiae KRE6 and SKN1: expression and physiological function.

    PubMed Central

    Mio, T; Yamada-Okabe, T; Yabe, T; Nakajima, T; Arisawa, M; Yamada-Okabe, H

    1997-01-01

    Cell wall beta-glucan in a pathogenic fungus, Candida albicans, is highly branched with beta-1,3 and beta-1,6 linkages. We have isolated the C. albicans cDNAs for KRE6 and SKN1, the genes required for beta-1,6-glucan synthesis in Saccharomyces cerevisiae. The results of Northern blot analysis revealed that C. albicans KRE6 was expressed at a higher level than SKN1 in the yeast phase, while SKN1 expression was strongly induced upon induction of hyphal formation. In addition, the C. albicans KRE6 and SKN1 mRNAs but not the actin mRNA were shortened during the yeast-hypha transition. Unlike S. cerevisiae, more than 50% of cell wall glucan was beta-1,6 linked in C. albicans. Neither beta-1,3-glucan nor beta-1,6-glucan was affected by the homozygous C. albicans skn1 delta null mutation. Although we never succeeded in generating the homozygous C. albicans kre6 delta null mutant, the hemizygous kre6 delta mutation decreased the KRE6 mRNA level by about 60% and also caused a more than 80% reduction of beta-1,6-glucan without affecting beta-1,3-glucan. The physiological function of KRE6 was further examined by studying gene regulation in C. albicans. When KRE6 transcription was suppressed by using the HEX1 promoter, C. albicans cells exhibited the partial defect in cell separation and increased susceptibility to Calcofluor White. These results demonstrate that KRE6 plays important roles in beta-1,6-glucan synthesis and budding in C. albicans. PMID:9079924

  10. A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

    PubMed Central

    Wing, S S; Bédard, N; Morales, C; Hingamp, P; Trasler, J

    1996-01-01

    The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins. PMID:8754804

  11. Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in Saccharomyces cerevisiae.

    PubMed

    Kumar, Navinder; Gaur, Deepika; Gupta, Arpit; Puri, Anuradhika; Sharma, Deepak

    2015-10-01

    The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

  12. Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae.

    PubMed Central

    Yamashiro, C T; Ebbole, D J; Lee, B U; Brown, R E; Bourland, C; Madi, L; Yanofsky, C

    1996-01-01

    The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10'-'hph). Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10'-'hph gene fusion. A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene. Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation). Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type. The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages. Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR. The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes. Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic. A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes. PMID:8887652

  13. The human homolog of S. cerevisiae CDC27, CDC27 Hs, is encoded by a highly conserved intronless gene present in multiple copies in the human genome

    SciTech Connect

    Devor, E.J.; Dill-Devor, R.M.

    1994-09-01

    We have obtained a number of unique sequences via PCR amplification of human genomic DNA using degenerate primers under low stringency (42{degrees}C). One of these, an 853 bp product, has been identified as a partial genomic sequence of the human homolog of the S. cerevisiae CDC27 gene, CDC27Hs (GenBank No. U00001). This gene, reported by Turgendreich et al. is also designated EST00556 from Adams et al. We have undertaken a more detailed examination of our sequence, MCP34N, and have found that: 1. the genomic sequence is nearly identical to CDC27Hs over its entire 853 bp length; 2. an MCP34N-specific PCR assay of several non-human primate species reveals amplification products in chimpanzee and gorilla genomes having greater than 90% sequence identity with CDC27Hs; and 3. an MCP34N-specific PCR assay of the BIOS hybrid cell line panel gives a discordancy pattern suggesting multiple loci. Based upon these data, we present the following initial characterization: 1. the complete MCP34N sequence identity with CDC27Hs indicates that the latter is encoded by an intronless gene; 2. CDC27Hs is highly conserved among higher primates; and 3. CDC27Hs is present in multiple copies in the human genome. These characteristics, taken together with those initially reported for CDC27Hs, suggest that this is an old gene that carries out an important but, as yet, unknown function in the human brain.

  14. Sequence of the pckA gene of Escherichia coli K-12: relevance to genetic and allosteric regulation and homology of E. coli phosphoenolpyruvate carboxykinase with the enzymes from Trypanosoma brucei and Saccharomyces cerevisiae.

    PubMed Central

    Medina, V; Pontarollo, R; Glaeske, D; Tabel, H; Goldie, H

    1990-01-01

    The sequence of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli K-12 and previous molecular weight determinations indicate that this allosteric enzyme is a monomer of Mr 51,316. The protein is homologous to ATP-dependent phosphoenolpyruvate carboxykinases from Trypanosoma brucei and Saccharomyces cerevisiae. A potential ATP binding site was conserved in all three sequences. A potential binding site for the allosteric activator, calcium, identified in the E. coli enzyme, was only partially conserved in T. brucei and S. cerevisiae, consistent with the observation that the enzymes from the latter organisms were not activated by calcium. The published sequence of the ompR and envZ genes from Salmonella typhimurium is followed by a partial sequence that is highly homologous to pckA from E. coli. The order of these genes and the direction of transcription of the presumptive S. typhimurium pckA gene are the same as those in E. coli. The potential calcium binding site of the E. coli enzyme is conserved in the partial predicted sequence of the S. typhimurium phosphoenolpyruvate carboxykinase, consistent with the observation that calcium activation of the S. typhimurium phosphoenolpyruvate carboxykinase is very similar to that observed for the E. coli enzyme. A pckA mRNA transcript was observed in stationary-phase cells but not in logarithmically growing cells. The mRNA start site was mapped relative to the sequence of the pckA structural gene. Images PMID:1701430

  15. The non-homologous end-joining pathway of S. cerevisiae works effectively in G1-phase cells, and religates cognate ends correctly and non-randomly.

    PubMed

    Gao, Shujuan; Honey, Sangeet; Futcher, Bruce; Grollman, Arthur P

    2016-06-01

    DNA double-strand breaks (DSBs) are potentially lethal lesions repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Homologous recombination preferentially reunites cognate broken ends. In contrast, non-homologous end-joining could ligate together any two ends, possibly generating dicentric or acentric fragments, leading to inviability. Here, we characterize the yeast NHEJ pathway in populations of pure G1 phase cells, where there is no possibility of repair using a homolog. We show that in G1 yeast cells, NHEJ is a highly effective repair pathway for gamma-ray induced breaks, even when many breaks are present. Pulsed-field gel analysis showed chromosome karyotypes following NHEJ repair of cells from populations with multiple breaks. The number of reciprocal translocations was surprisingly low, perhaps zero, suggesting that NHEJ preferentially re-ligates the "correct" broken ends instead of randomly-chosen ends. Although we do not know the mechanism, the preferential correct ligation is consistent with the idea that broken ends are continuously held together by protein-protein interactions or by larger scale chromatin structure. PMID:27130982

  16. FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

    PubMed

    Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S; Lu, Ling

    2014-02-01

    Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

  17. FigA, a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae, Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

    PubMed Central

    Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S.

    2014-01-01

    Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca2+ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development. PMID:24376003

  18. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    PubMed

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

  19. The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

    PubMed Central

    Johnston, L H; Eberly, S L; Chapman, J W; Araki, H; Sugino, A

    1990-01-01

    Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division. Images PMID:2181271

  20. cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B.

    PubMed Central

    Ishiguro, J; Saitou, A; Durán, A; Ribas, J C

    1997-01-01

    The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis. PMID:9401022

  1. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

    PubMed

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

    2015-04-01

    The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus.

  2. Increase in dual specificity phosphatase 1, TGF-beta stimulated gene 22, domain family protein 3 and Luc7 homolog (S. cerevisiae)-like messenger RNA after mechanical asphyxiation in the mouse lung.

    PubMed

    Takahashi, Hiroyuki; Ikematsu, Kazuya; Tsuda, Ryouichi; Nakasono, Ichiro

    2009-07-01

    We investigated the transcriptome profile of mechanical asphyxia and decapitation at 60 min after death using serial analysis of gene expression. After comparing the results, 11 genes were significantly increased by the mechanical asphyxia treatment in the mouse lung. Of those genes, quantitative real-time PCR revealed that dual specificity phosphatase 1 (Dusp1), TGF-beta stimulated gene 22, domain family protein 3 (TSC22d3) and Luc7 homolog (Saccharomyces cerevisiae)-like (Luc7l) after asphyxia were more significantly increased than those after decapitation. Dusp1 inactivated mitogen activated protein kinase, which functions in cell proliferation. However, the consumption of oxygen had a disadvantageous effect on survival, because tissue or cells were not able to produce energy by internal respiration under the suddenly hypoxic condition following asphyxia. The increased transcripts of Dusp1 following asphyxia suppressed oxygen consumption. TSC22d3 was isolated as a TGF-beta-inducible gene and it is also identified as a glucocorticoid (GC)-induced leucine zipper (GILZ). GC was released from the adrenal gland via HPA axis under the hypoxic condition. Especially in acute suffocation, GC rapidly increased. Therefore, the increase in TSC22d3 may be induced by the increased GC following asphyxia. We were unable to clarify the Luc7l increase, because there are no reports in relation to asphyxia. In addition, GILZ mediates the antiproliferative activity of glucocorticoids. We thought that the increasing TSC22d3 may lead to the suppression of oxygen consumption to avoid wasting energy, as in proliferation, the same as the increase in Dusp1. Our data indicated that the determination of the protein product level in the lung could help in diagnosing asphyxia. In addition, these data may contribute to revealing the patho-physiology of asphyxia and to help diagnose asphyxia, including hanging. PMID:19364672

  3. Alcohol homologation

    DOEpatents

    Wegman, Richard W.; Moloy, Kenneth G.

    1988-01-01

    A process for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  4. Alcohol homologation

    DOEpatents

    Wegman, R.W.; Moloy, K.G.

    1988-02-23

    A process is described for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

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

    PubMed

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

    2006-07-25

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

  6. Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition.

    PubMed

    Kaur, Hardeep; Datt, Manish; Ekka, Mary Krishna; Mittal, Monica; Singh, Appu Kumar; Kumaran, Sangaralingam

    2011-02-01

    Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction. Dug1p exists as dimer and the stoichiometry of peptide-Dug1p complex is 2:1 indicating each monomer in the dimer binds to one peptide. Thermodynamic studies indicate that the free energy change for Dug1p-peptide complex formation is similar (▵G(bind) ∼ -7.0 kcal/mol) for a variety of peptides of different composition and length (22 peptides). Three-dimensional model of Dug1p is constructed and docking of peptides to the modeled structure suggests that hydrogen bonding to active site residues (E172, E171, and D137) lock the N-terminal of the peptide into the binding site. Dug1p recognizes peptides in a metal independent manner and peptide binding is not sensitive to salts (dlogK/dlog[salt] ∼ 0) over a range of [NaCl] (0.02-0.5 M), [ZnCl(2)], and [MnCl(2)] (0-0.5 mM). Our results indicate that promiscuity in peptide binding results from the locking of peptide N-terminus into the active site. These observations were supported by our competitive inhibition activity assays. Dug1p activity towards Cys-Gly peptide is significantly reduced (∼ 70%) in the presence of Glu-Cys-Gly. Therefore, Dug1p can recognize a variety of oligopeptides, but has evolved with post-binding screening potential to hydrolyze Cys-Gly peptides selectively.

  7. Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR).

    PubMed

    Chandran, Sunil; Shapland, Elaine

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the yeast homologous recombination (YHR). The YHR method utilizes overlapping DNA parts that are assembled together by Saccharomyces cerevisiae via homologous recombination between designed overlapping regions. Using this method, we have successfully assembled up to 12 DNA parts in a single reaction. PMID:27671941

  8. The hexose transporter family of Saccharomyces cerevisiae.

    PubMed

    Kruckeberg, A L

    1996-11-01

    Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web.

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

    PubMed

    Alcalde, Miguel

    2010-01-01

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

  10. Nitrogenase and Homologs

    PubMed Central

    2014-01-01

    Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes. PMID:25491285

  11. Homological stabilizer codes

    SciTech Connect

    Anderson, Jonas T.

    2013-03-15

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev's toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev's toric code or to the topological color codes. - Highlights: Black-Right-Pointing-Pointer We show that Kitaev's toric codes are equivalent to homological stabilizer codes on 4-valent graphs. Black-Right-Pointing-Pointer We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. Black-Right-Pointing-Pointer We find and classify all 2D homological stabilizer codes. Black-Right-Pointing-Pointer We find optimal codes among the homological stabilizer codes.

  12. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

    PubMed Central

    Schiestl, R H; Petes, T D

    1991-01-01

    DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends of BamHI fragments, this results indicates that four base pairs of homology are sufficient for some types of mitotic recombination. Images PMID:1881899

  13. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  14. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  15. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  17. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  18. Homology, convergence and parallelism.

    PubMed

    Ghiselin, Michael T

    2016-01-01

    Homology is a relation of correspondence between parts of parts of larger wholes. It is used when tracking objects of interest through space and time and in the context of explanatory historical narratives. Homologues can be traced through a genealogical nexus back to a common ancestral precursor. Homology being a transitive relation, homologues remain homologous however much they may come to differ. Analogy is a relationship of correspondence between parts of members of classes having no relationship of common ancestry. Although homology is often treated as an alternative to convergence, the latter is not a kind of correspondence: rather, it is one of a class of processes that also includes divergence and parallelism. These often give rise to misleading appearances (homoplasies). Parallelism can be particularly hard to detect, especially when not accompanied by divergences in some parts of the body. PMID:26598721

  19. Homology, limbs, and genitalia.

    PubMed

    Minelli, Alessandro

    2002-01-01

    Similarities in genetic control between the main body axis and its appendages have been generally explained in terms of genetic co-option. In particular, arthropod and vertebrate appendages have been explained to invoke a common ancestor already provided with patterned body outgrowths or independent recruitment in limb patterning of genes or genetic cassettes originally used for purposes other than axis patterning. An alternative explanation is that body appendages, including genitalia, are evolutionarily divergent duplicates (paramorphs) of the main body axis. However, are all metazoan limbs and genitalia homologous? The concept of body appendages as paramorphs of the main body axis eliminates the requirement for the last common ancestor of limb-bearing animals to have been provided with limbs. Moreover, the possibility for an animal to express complex organs ectopically demonstrates that positional and special homology may be ontogenetically and evolutionarily uncoupled. To assess the homology of animal genitalia, we need to take into account three different sets of mechanisms, all contributing to their positional and/or special homology and respectively involved (1) in the patterning of themain body axis, (2) in axis duplication, followed by limb patterning mechanisms diverging away from those still patterning the main body axis (axis paramorphism), and (3) in controlling the specification of sexual/genital features, which often, but not necessarily, come into play by modifying already developed and patterned body appendages. This analysis demonstrates that a combinatorial approach to homology helps disentangling phylogenetic and ontogenetic layers of homology.

  20. Mobilomics in Saccharomyces cerevisiae strains

    PubMed Central

    2013-01-01

    Background Mobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus–like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated. Results Our approach proposes to exploit the high similarity among homologous chromosomes of different strains of the same species, following a progressive comparative genomics philosophy. We introduce a software tool based on our new fast algorithm, called regender, which is able to identify the conserved regions between chromosomes. Our case study is represented by a unique recently available dataset of 39 different strains of S.cerevisiae, which regender is able to compare in few minutes. By exploring the non–conserved regions, where MGEs are mainly retrotransposons called Tys, and marking the candidate Tys based on their length, we are able to locate a priori and automatically all the already known Tys and map all the putative Tys in all the strains. The remaining putative mobile elements (PMEs) emerging from this intra–specific comparison are sharp markers of inter–specific evolution: indeed, many events of non–conservation among different yeast strains correspond to PMEs. A clustering based on the presence/absence of the candidate Tys in the strains suggests an evolutionary interconnection that is very similar to classic phylogenetic trees based on SNPs analysis, even though it is computed without using phylogenetic information. Conclusions The case study indicates that the proposed methodology brings two major advantages: (a) it does not require any template sequence for the wanted MGEs and (b) it can be applied to

  1. Innovation by homologous recombination.

    PubMed

    Trudeau, Devin L; Smith, Matthew A; Arnold, Frances H

    2013-12-01

    Swapping fragments among protein homologs can produce chimeric proteins with a wide range of properties, including properties not exhibited by the parents. Computational methods that use information from structures and sequence alignments have been used to design highly functional chimeras and chimera libraries. Recombination has generated proteins with diverse thermostability and mechanical stability, enzyme substrate specificity, and optogenetic properties. Linear regression, Gaussian processes, and support vector machine learning have been used to model sequence-function relationships and predict useful chimeras. These approaches enable engineering of protein chimeras with desired functions, as well as elucidation of the structural basis for these functions.

  2. Homology, homoplasy, novelty, and behavior.

    PubMed

    Hall, Brian K

    2013-01-01

    Richard Owen coined the modern definition of homology in 1843. Owen's conception of homology was pre-evolutionary, nontransformative (homology maintained basic plans or archetypes), and applied to the fully formed structures of animals. I sketch out the transition to an evolutionary approach to homology in which all classes of similarity are interpreted against the single branching tree of life, and outline the evidence for the application of homology across all levels and features of the biological hierarchy, including behavior. Owen contrasted homology with analogy. While this is not incorrect it is a pre-evolutionary contrast. Lankester [Lankester [1870] Journal of Natural History, 6 (31), 34-43] proposed homoplasy as the class of homology applicable to features formed by independent evolution. Today we identify homology, convergence, parallelism, and novelties as patterns of evolutionary change. A central issue in homology [Owen [1843] Lectures on comparative anatomy and physiology of the invertebrate animals, delivered at the Royal College of Surgeons in 1843. London: Longman, Brown, Green & Longmans] has been whether homology of features-the "same" portion of the brain in different species, for example-depends upon those features sharing common developmental pathways. Owen did not require this criterion, although he observed that homologues often do share developmental pathways (and we now know, often share gene pathways). A similar situation has been explored in the study of behavior, especially whether behaviors must share a common structural, developmental, neural, or genetic basis to be classified as homologous. However, and importantly, development and genes evolve. As shown with both theory and examples, morphological and behavioral features of the phenotype can be homologized as structural or behavioral homologues, respectively, even when their developmental or genetic bases differ (are not homologous). PMID:22711423

  3. Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

    PubMed

    Anderson, G J; Lesuisse, E; Dancis, A; Roman, D G; Labbe, P; Klausner, R D

    We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.

  4. Initiation of recombination in Saccharomyces cerevisiae haploid meiosis.

    PubMed Central

    De Massy, B; Baudat, F; Nicolas, A

    1994-01-01

    In most eukaryotes during prophase I of meiosis, homologous chromosomes pair and recombine by coordinated molecular and cellular processes. To directly test whether or not the early steps of the initiation of recombination depend on the presence of a homologous chromosome, we have examined the formation and processing of DNA double-strand breaks (DSBs, the earliest physical landmark of recombination initiation) in various haploid Saccharomyces cerevisiae strains capable of entering meiosis. We find that DSBs occur in haploid meiosis, showing that the presence of a homolog is not required for DSB formation. DSBs occur at the same positions in haploid and diploid meioses. However, these two types of meiosis exhibit subtle differences with respect to the timing of formation and levels of DSBs. In haploid meiosis, a slower rate of DSB formation and a reduction in the frequency of DSB (at one of the three sites analyzed) were observed. These results might indicate that interactions between homologs play a role in the formation of meiotic DSBs. Furthermore, haploid strains exhibit a pronounced delay in the disappearance of meiotic DSBs compared to diploid strains, which suggests that sister chromatid interactions for DSB repair are inhibited in haploid meiosis. Images PMID:7991559

  5. Characterization of cDNA encoding mouse homolog of fission yeast dhp1+ gene: structural and functional conservation.

    PubMed Central

    Shobuike, T; Sugano, S; Yamashita, T; Ikeda, H

    1995-01-01

    The dhp1+ gene of Schizosaccharomyces pombe is a homolog of Saccharomyces cerevisiae HKE1/RAT1/TAP1 gene that is involved in RNA metabolism such as RNA trafficking and RNA synthesis. dhp1+ is also related to S. cerevisiae DST2 (SEP1) that encodes a DNA strand exchange protein required for sporulation and homologous recombination in S.cerevisiae. We isolated several clones of Dhm1, a mouse homolog of dhp1+, from mouse spermatocyte cDNA library and determined its nucleotide sequence. The Dhm1 gene consists of an open reading frame predicting a protein with 947 amino acids and molecular weight of 107,955. Northern blot analysis revealed that Dhm1 is transcribed at high level in testis, liver and kidney. The predicted product of Dhm1 (Dhm1p) has a significant homology with Dhp1p, Hke1p/Rat1p/Tap1p and Dst2p. In particular, Dhm1p, Dhp1p and Hke1p/Rat1p/Tap1p share strong similarity at the two regions of their N- and C-terminal parts. The Dhm1 gene on a multicopy plasmid rescued the temperature-sensitivity of dhp1ts and lethality of dhp1 null mutation, suggesting that Dhm1 is a mouse homolog of S.pombe dhp1+ and functions similarly in mouse as dhp1+. Images PMID:7885830

  6. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

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

  7. Evolving the Concept of Homology

    ERIC Educational Resources Information Center

    Naples, Virginia L.; Miller, Jon S.

    2009-01-01

    Understanding homology is fundamental to learning about evolution. The present study shows an exercise that can be varied in complexity, for which students compile research illustrating the fate of homologous fish skull elements, and assemble a mural to serve as a learning aid. The skull of the most primitive living Actinopterygian (bony fish),…

  8. Object-oriented persistent homology

    NASA Astrophysics Data System (ADS)

    Wang, Bao; Wei, Guo-Wei

    2016-01-01

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  9. The semaphorontic view of homology

    PubMed Central

    Assis, Leandro C.S.; Rieppel, Olivier

    2015-01-01

    ABSTRACT The relation of homology is generally characterized as an identity relation, or alternatively as a correspondence relation, both of which are transitive. We use the example of the ontogenetic development and evolutionary origin of the gnathostome jaw to discuss identity and transitivity of the homology relation under the transformationist and emergentist paradigms respectively. Token identity and consequent transitivity of homology relations are shown to be requirements that are too strong to allow the origin of genuine evolutionary novelties. We consequently introduce the concept of compositional identity that is grounded in relations prevailing between parts (organs and organ systems) of a whole (organism). We recognize an ontogenetic identity of parts within a whole throughout the sequence of successive developmental stages of those parts: this is an intra‐organismal character identity maintained throughout developmental trajectory. Correspondingly, we recognize a phylogenetic identity of homologous parts within two or more organisms of different species: this is an inter‐species character identity maintained throughout evolutionary trajectory. These different dimensions of character identity—ontogenetic (through development) and phylogenetic (via shared evolutionary history)—break the transitivity of homology relations. Under the transformationist paradigm, the relation of homology reigns over the entire character (‐state) transformation series, and thus encompasses the plesiomorphic as well as the apomorphic condition of form. In contrast, genuine evolutionary novelties originate not through transformation of ancestral characters (‐states), but instead through deviating developmental trajectories that result in alternate characters. Under the emergentist paradigm, homology is thus synonymous with synapomorphy. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 578–587, 2015. © 2015 The Authors. Journal of Experimental Zoology Part B: Molecular and

  10. PET genes of Saccharomyces cerevisiae.

    PubMed Central

    Tzagoloff, A; Dieckmann, C L

    1990-01-01

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

  11. Saccharomyces cerevisiae-based system for studying clustered DNA damages

    SciTech Connect

    Moscariello, M.M.; Sutherland, B.

    2010-08-01

    DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.

  12. CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    PubMed Central

    Sasano, Yu; Nagasawa, Koki; Kaboli, Saeed; Sugiyama, Minetaka; Harashima, Satoshi

    2016-01-01

    PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function. PMID:27530680

  13. Purification and Characterization of Put1p from Saccharomyces cerevisiae

    PubMed Central

    Wanduragala, Srimevan; Sanyal, Nikhilesh; Liang, Xinwen; Becker, Donald F.

    2010-01-01

    In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH)1 enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A Km value of 36 mM proline and a kcat = 27 s−1 were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ1) as an electron acceptor with a kcat = 9.6 s−1 and a Km of 33 µM for CoQ1. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast. PMID:20450881

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

    PubMed

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

    2016-06-01

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

  15. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  16. Sequence analysis of 203 kilobases from Saccharomyces cerevisiae chromosome VII.

    PubMed

    Rieger, M; Brückner, M; Schäfer, M; Müller-Auer, S

    1997-09-15

    The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15.8 kb starts approximately 75 kb from the left telomere and is bordered by the SK18 chromosomal marker. The second fragment covers the 72.6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70.6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced. We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction. A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome. Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs. Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence. The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. PMID:9290212

  17. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p

    PubMed Central

    Li, Li; Lipke, Peter N.; Dranginis, Anne M.

    2016-01-01

    ABSTRACT FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the

  18. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p.

    PubMed

    Barua, Subit; Li, Li; Lipke, Peter N; Dranginis, Anne M

    2016-01-01

    FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the expression of

  19. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  20. Saccharomyces cerevisiae osteomyelitis in an immunocompetent baker.

    PubMed

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

    2016-01-01

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

  1. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

    PubMed Central

    Sweetser, D; Nonet, M; Young, R A

    1987-01-01

    Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases. PMID:3547406

  2. Cadmium biosorption by Saccharomyces cerevisiae

    SciTech Connect

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

    1993-04-01

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

  3. Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

    PubMed Central

    Kaishima, Misato; Ishii, Jun; Matsuno, Toshihide; Fukuda, Nobuo; Kondo, Akihiko

    2016-01-01

    Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry. PMID:27782154

  4. Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae

    PubMed Central

    Haber, James E.

    2012-01-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break. PMID:22555442

  5. Mating-type genes and MAT switching in Saccharomyces cerevisiae.

    PubMed

    Haber, James E

    2012-05-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break.

  6. A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae.

    PubMed

    Zelter, Alex; Bencina, Mojca; Bowman, Barry J; Yarden, Oded; Read, Nick D

    2004-09-01

    A large number of Ca2+ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca2+ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca2+ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca2+ -permeable channels in each species; 9, 12, and 5 Ca2+/cation-ATPases; eight, six, and four Ca2+ -exchangers; four, four, and two phospholipase C's; one calmodulin in each species; and 23, 21, and 29 Ca2+/calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca2+ from intracellular stores, and in the sensing of extracellular Ca2+, in animal and plant cells, were not identified. The greater complexity of the Ca2+ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource (http://www.fungalcell.org/fdf/) of all Ca2+ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.

  7. ISHAN: sequence homology analysis package.

    PubMed

    Shil, Pratip; Dudani, Niraj; Vidyasagar, Pandit B

    2006-01-01

    Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention. PMID:17274766

  8. A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae

    PubMed Central

    Chu, Daniel B.; Burgess, Sean M.

    2016-01-01

    Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

  9. A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae.

    PubMed

    Chu, Daniel B; Burgess, Sean M

    2016-03-01

    Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

  10. Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification.

    PubMed

    Luhe, Annette Lin; Tan, Lily; Wu, Jinchuan; Zhao, Hua

    2011-05-01

    Saccharomyces cerevisiae was transformed for higher ethanol tolerance by error-prone whole genome amplification. The resulting PCR products were transformed back to the parental strain for homologous recombination to create a library of mutants with the perturbed genomic networks. A few rounds of transformation led to the isolation of mutants that grew in 9% (v/v) ethanol and 100 g glucose l(-1) compared to untransformed yeast which grew only at 6% (v/v) ethanol and 100 g glucose l(-1).

  11. The essential helicase gene RAD3 suppresses short-sequence recombination in Saccharomyces cerevisiae.

    PubMed Central

    Bailis, A M; Maines, S; Negritto, M T

    1995-01-01

    We have isolated an allele of the essential DNA repair and transcription gene RAD3 that relaxes the restriction against recombination between short DNA sequences in Saccharomyces cerevisiae. Double-strand break repair and gene replacement events requiring recombination between short identical or mismatched sequences were stimulated in the rad3-G595R mutant cells. We also observed an increase in the physical stability of double-strand breaks in the rad3-G595R mutant cells. These results suggest that the RAD3 gene suppresses recombination involving short homologous sequences by promoting the degradation of the ends of broken DNA molecules. PMID:7623796

  12. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

    PubMed Central

    Duncan, K; Edwards, R M; Coggins, J R

    1987-01-01

    The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes. PMID:2825635

  13. Chromosomal Assignment of Mutations by Specific Chromosome Loss in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Wakem, L. P.; Sherman, F.

    1990-01-01

    Yeast 2-μm plasmids were integrated near the centromere of a different chromosome in each of 16 cir(0) mapping strains of Saccharomyces cerevisiae. The specific chromosomes containing the integrated 2-μm plasmid DNA were lost at a high frequency after crossing the cir(0) strains to cir(+) strains. A recessive mutation in a cir(+) strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-μm plasmid and the unmapped mutation on homologous chromosomes. PMID:2199315

  14. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

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

  15. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

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

  16. Chronological aging in Saccharomyces cerevisiae.

    PubMed

    Longo, Valter D; Fabrizio, Paola

    2012-01-01

    The two paradigms to study aging in Saccharomyces cerevisiae are the chronological life span (CLS) and the replicative life span (RLS). The chronological life span is a measure of the mean and maximum survival time of non-dividing yeast populations while the replicative life span is based on the mean and maximum number of daughter cells generated by an individual mother cell before cell division stops irreversibly. Here we review the principal discoveries associated with yeast chronological aging and how they are contributing to the understanding of the aging process and of the molecular mechanisms that may lead to healthy aging in mammals. We will focus on the mechanisms of life span regulation by the Tor/Sch9 and the Ras/adenylate Ras/adenylate cyclase/PKA pathways with particular emphasis on those implicating age-dependent oxidative oxidative stress stress and DNA damage/repair.

  17. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

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

  18. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Edlarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  19. Flavour formation in fungi: characterisation of KlAtf, the Kluyveromyces lactis orthologue of the Saccharomyces cerevisiae alcohol acetyltransferases Atf1 and Atf2.

    PubMed

    Van Laere, Stijn D M; Saerens, Sofie M G; Verstrepen, Kevin J; Van Dijck, Patrick; Thevelein, Johan M; Delvaux, Freddy R

    2008-04-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers' yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.

  20. Establishing homologies in protein sequences

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.; Barker, W. C.; Hunt, L. T.

    1983-01-01

    Computer-based statistical techniques used to determine homologies between proteins occurring in different species are reviewed. The technique is based on comparison of two protein sequences, either by relating all segments of a given length in one sequence to all segments of the second or by finding the best alignment of the two sequences. Approaches discussed include selection using printed tabulations, identification of very similar sequences, and computer searches of a database. The use of the SEARCH, RELATE, and ALIGN programs (Dayhoff, 1979) is explained; sample data are presented in graphs, diagrams, and tables and the construction of scoring matrices is considered.

  1. Homologous gene replacement in Physarum

    SciTech Connect

    Burland, T.G.; Pallotta, D.

    1995-01-01

    The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD{Delta}1, of the ardD actin gene; ardD{Delta}1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD{sup +} with ardD{Delta}1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD{Delta}1 in addition to ardD{sup +}, but in two (5%), ardD{sup +} was replaced by a single copy of ardD{Delta}1. This is the first example of homologous gene replacement in Physarum. ardD{Delta}1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD{Delta}l on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene. 38 refs., 6 figs., 1 tab.

  2. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    SciTech Connect

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  3. Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

    PubMed Central

    Yasugi, T; Howley, P M

    1996-01-01

    Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression. PMID:8668529

  4. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein.

    PubMed

    Vojtek, A B; Cooper, J A

    1993-07-01

    CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

  5. SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes.

    PubMed Central

    Gerst, J E; Rodgers, L; Riggs, M; Wigler, M

    1992-01-01

    SNC1, a gene from the yeast Saccharomyces cerevisiae, encodes a homolog of vertebrate synaptic vesicle-associated membrane proteins (VAMPs) or synaptobrevins. SNC1 was isolated by its ability to suppress the loss of CAP function in S. cerevisiae strains possessing an activated allele of RAS2. CAP is a component of the RAS-responsive S. cerevisiae adenylyl cyclase complex. The N-terminal domain of CAP is required for full cellular responsiveness to activated RAS proteins. The C-terminal domain of CAP is required for normal cellular morphology and responsiveness to nutrient extremes. Multicopy plasmids expressing SNC1 suppress only the loss of the C-terminal functions of CAP and only in the presence of activated RAS2. Images PMID:1316605

  6. Improvement of lactic acid production in Saccharomyces cerevisiae by a deletion of ssb1.

    PubMed

    Lee, Jinsuk J; Crook, Nathan; Sun, Jie; Alper, Hal S

    2016-01-01

    Polylactic acid (PLA) is an important renewable polymer, but current processes for producing its precursor, lactic acid, suffer from process inefficiencies related to the use of bacterial hosts. Therefore, improving the capacity of Saccharomyces cerevisiae to produce lactic acid is a promising approach to improve industrial production of lactic acid. As one such improvement required, the lactic acid tolerance of yeast must be significantly increased. To enable improved tolerance, we employed an RNAi-mediated genome-wide expression knockdown approach as a means to rapidly identify potential genetic targets. In this approach, several gene knockdown targets were identified which confer increased acid tolerance to S. cerevisiae BY4741, of which knockdown of the ribosome-associated chaperone SSB1 conferred the highest increase (52%). This target was then transferred into a lactic acid-overproducing strain of S. cerevisiae CEN.PK in the form of a knockout and the resulting strain demonstrated up to 33% increased cell growth, 58% increased glucose consumption, and 60% increased L-lactic acid production. As SSB1 contains a close functional homolog SSB2 in yeast, this result was counterintuitive and may point to as-yet-undefined functional differences between SSB1 and SSB2 related to lactic acid production. The final strain produced over 50 g/L of lactic acid in under 60 h of fermentation.

  7. Persistent homology and string vacua

    NASA Astrophysics Data System (ADS)

    Cirafici, Michele

    2016-03-01

    We use methods from topological data analysis to study the topological features of certain distributions of string vacua. Topological data analysis is a multi-scale approach used to analyze the topological features of a dataset by identifying which homological characteristics persist over a long range of scales. We apply these techniques in several contexts. We analyze {N}=2 vacua by focusing on certain distributions of Calabi-Yau varieties and Landau-Ginzburg models. We then turn to flux compactifications and discuss how we can use topological data analysis to extract physical information. Finally we apply these techniques to certain phenomenologically realistic heterotic models. We discuss the possibility of characterizing string vacua using the topological properties of their distributions.

  8. Gene Duplication in SACCHAROMYCES CEREVISIAE

    PubMed Central

    Hansche, P. E.; Beres, V.; Lange, P.

    1978-01-01

    Five indepdendent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found. PMID:348562

  9. Human DNA ligase I cDNA: Cloning and functional expression in Saccharomyces cerevisiae

    SciTech Connect

    Barnes, D.E.; Kodama, Kenichi; Tomkinson, A.E.; Lindahl, T.; Lasko, D.D. ); Johnston, L.H. )

    1990-09-01

    Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomuces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

  10. Primers-4-Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae.

    PubMed

    Yofe, Ido; Schuldiner, Maya

    2014-02-01

    The budding yeast Saccharomyces cerevisiae is a key model organism of functional genomics, due to its ease and speed of genetic manipulations. In fact, in this yeast, the requirement for homologous sequences for recombination purposes is so small that 40 base pairs (bp) are sufficient. Hence, an enormous variety of genetic manipulations can be performed by simply planning primers with the correct homology, using a defined set of transformation plasmids. Although designing primers for yeast transformations and for the verification of their correct insertion is a common task in all yeast laboratories, primer planning is usually done manually and a tool that would enable easy, automated primer planning for the yeast research community is still lacking. Here we introduce Primers-4-Yeast, a web tool that allows primers to be designed in batches for S. cerevisiae gene-targeting transformations, and for the validation of correct insertions. This novel tool enables fast, automated, accurate primer planning for large sets of genes, introduces consistency in primer planning and is therefore suggested to serve as a standard in yeast research. Primers-4-Yeast is available at: http://www.weizmann.ac.il/Primers-4-Yeast

  11. Nonsense-mediated decay regulates key components of homologous recombination

    PubMed Central

    Janke, Ryan; Kong, Jeremy; Braberg, Hannes; Cantin, Greg; Yates, John R.; Krogan, Nevan J.; Heyer, Wolf-Dietrich

    2016-01-01

    Cells frequently experience DNA damage that requires repair by homologous recombination (HR). Proteins involved in HR are carefully coordinated to ensure proper and efficient repair without interfering with normal cellular processes. In Saccharomyces cerevisiae, Rad55 functions in the early steps of HR and is regulated in response to DNA damage through phosphorylation by the Mec1 and Rad53 kinases of the DNA damage response. To further identify regulatory processes that target HR, we performed a high-throughput genetic interaction screen with RAD55 phosphorylation site mutants. Genes involved in the mRNA quality control process, nonsense-mediated decay (NMD), were found to genetically interact with rad55 phospho-site mutants. Further characterization revealed that RAD55 transcript and protein levels are regulated by NMD. Regulation of HR by NMD extends to multiple targets beyond RAD55, including RAD51, RAD54 and RAD57. Finally, we demonstrate that loss of NMD results in an increase in recombination rates and resistance to the DNA damaging agent methyl methanesulfonate, suggesting this pathway negatively regulates HR under normal growth conditions. PMID:27001511

  12. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    PubMed

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  13. Biosynthesis of silver nanoparticles using Saccharomyces cerevisiae.

    PubMed

    Korbekandi, Hassan; Mohseni, Soudabeh; Mardani Jouneghani, Rasoul; Pourhossein, Meraj; Iravani, Siavash

    2016-01-01

    The objectives of this study were the biosynthesis of silver nanoparticles (NPs) by biotransformations using Saccharomyces cerevisiae and analysis of the sizes and shapes of the NPs produced. Dried and freshly cultured S. cerevisiae were used as the biocatalyst. Dried yeast synthesized few NPs, but freshly cultured yeast produced a large amount of them. Silver NPs were spherical, 2-20 nm in diameter, and the NPs with the size of 5.4 nm were the most frequent ones. NPs were seen inside the cells, within the cell membrane, attached to the cell membrane during the exocytosis, and outside of the cells.

  14. Buoyancy instability of homologous implosions

    NASA Astrophysics Data System (ADS)

    Johnson, Bryan

    2015-11-01

    Hot spot turbulence is a potential contributor to yield degradation in inertial confinement fusion (ICF) capsules, although its origin, if present, remains unclear. In this work, a perturbation analysis is performed of an analytical homologous solution that mimics the hot spot and surrounding cold fuel during the late stages of an ICF implosion. It is shown that the flow is governed by the Schwarzschild criterion for buoyant stability, and that during stagnation, short wavelength entropy and vorticity fluctuations amplify by a factor exp (π |N0 | ts) , where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. This amplification factor is exponentially sensitive to mean flow gradients and varies from 103-107 for realistic gradients. Comparisons are made with a Lagrangian hydrodynamics code, and it is found that a numerical resolution of ~ 30 zones per wavelength is required to capture the evolution of vorticity accurately. This translates to an angular resolution of ~(12 / l) ∘ , or ~ 0 .1° to resolve the fastest growing modes (Legendre mode l > 100).

  15. Homology among bacterial catalase genes.

    PubMed

    Switala, J; Triggs-Raine, B L; Loewen, P C

    1990-10-01

    Catalase activities in crude extracts of exponential and stationary phase cultures of various bacteria were visualized following gel electrophoresis for comparison with the enzymes from Escherichia coli. Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Klebsiella pneumoniae, and Salmonella typhimurium exhibited patterns of catalase activity similar to E. coli, including bifunctional HPI-like bands and a monofunctional HPII-like band. Proteus mirabilis, Erwinia carotovora, and Serratia marcescens contained a single band of monofunctional catalase with a mobility intermediate between the HPI-like and HPII-like bands. The cloned genes for catalases HPI (katG) and HPII (katE) from E. coli were used as probes in Southern hybridization analyses for homologous sequences in genomic DNA of the same bacteria. katG was found to hybridize with fragments from C. freudii, Ent. aerogenes, Sal. typhimurium, and K. pneumoniae but not at all with Ed. tarda, P. mirabilis, S. marcesens, or Er. carotovora. katE hybridized with C. freundii and K. pneumoniae DNAs and not with the other bacterial DNAs.

  16. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  17. Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

    PubMed Central

    Dornfeld, K. J.; Livingston, D. M.

    1992-01-01

    Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles. PMID:1644271

  18. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

  19. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

    PubMed

    Stovicek, Vratislav; Borja, Gheorghe M; Forster, Jochen; Borodina, Irina

    2015-11-01

    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.

  20. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    SciTech Connect

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-22

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  1. Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product

    PubMed Central

    Ikushima, Shigehito; Zhao, Yu; Boeke, Jef D.

    2015-01-01

    Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule (at micromolar concentrations) for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor. PMID:26206350

  2. Characterization of the Saccharomyces cerevisiae FMS1 gene related to Candida albicans corticosteroid-binding protein 1.

    PubMed

    Joets, J; Pousset, D; Marcireau, C; Karst, F

    1996-07-31

    In order to investigate ergosterol metabolism in S. cerevisiae we studied the CM8 mutant strain defective in the regulation of this pathway. A genomic multicopy library was screened to reverse the CM8 phenotype. This allowed us to characterize a new gene, FMS1, which relieves mutant phenotype by extragenic functional complementation. FMS1 may encode a 508 amino-acid protein. The predicted protein shares 35% identity with Cbp1p, a Candida albicans corticosteroid binding-protein. Fms1p also shows a weaker homology with monoamine oxidases. The construction of a FMS1 null-allele yeast strain demonstrated that this gene is not essential for yeast in normal usual laboratory culture conditions. The existence of a gene related to CBP1 of C. albicans in S. cerevisiae strongly suggests a possible function of steroid-binding proteins in yeast general physiology rather than in a process related to pathogenicity.

  3. Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

    PubMed

    Becker-Kettern, Julia; Paczia, Nicole; Conrotte, Jean-François; Kay, Daniel P; Guignard, Cédric; Jung, Paul P; Linster, Carole L

    2016-03-18

    The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.

  4. Engineer Sccharomyces cerevisiae for consolidated bioprocessing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The current commercial biofuel production is based on a two-stage process of enzymatic treatment to degrade starch to fermentable sugar, followed by yeast fermentation of the sugar to ethanol. An attractive alternative would be to engineer Saccharomyces cerevisiae for cell-based saccharification an...

  5. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  6. Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

    PubMed Central

    Jin, Yong-Su; Alper, Hal; Yang, Yea-Tyng; Stephanopoulos, Gregory

    2005-01-01

    We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae. PMID:16332810

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

    SciTech Connect

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

    1998-08-01

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

  8. Homology-Independent Metrics for Comparative Genomics

    PubMed Central

    Coutinho, Tarcisio José Domingos; Franco, Glória Regina; Lobo, Francisco Pereira

    2015-01-01

    A mainstream procedure to analyze the wealth of genomic data available nowadays is the detection of homologous regions shared across genomes, followed by the extraction of biological information from the patterns of conservation and variation observed in such regions. Although of pivotal importance, comparative genomic procedures that rely on homology inference are obviously not applicable if no homologous regions are detectable. This fact excludes a considerable portion of “genomic dark matter” with no significant similarity — and, consequently, no inferred homology to any other known sequence — from several downstream comparative genomic methods. In this review we compile several sequence metrics that do not rely on homology inference and can be used to compare nucleotide sequences and extract biologically meaningful information from them. These metrics comprise several compositional parameters calculated from sequence data alone, such as GC content, dinucleotide odds ratio, and several codon bias metrics. They also share other interesting properties, such as pervasiveness (patterns persist on smaller scales) and phylogenetic signal. We also cite examples where these homology-independent metrics have been successfully applied to support several bioinformatics challenges, such as taxonomic classification of biological sequences without homology inference. They where also used to detect higher-order patterns of interactions in biological systems, ranging from detecting coevolutionary trends between the genomes of viruses and their hosts to characterization of gene pools of entire microbial communities. We argue that, if correctly understood and applied, homology-independent metrics can add important layers of biological information in comparative genomic studies without prior homology inference. PMID:26029354

  9. Buoyancy instability of homologous implosions

    SciTech Connect

    Johnson, B. M.

    2015-06-15

    With this study, I consider the hydrodynamic stability of imploding ideal gases as an idealized model for inertial confinement fusion capsules, sonoluminescent bubbles and the gravitational collapse of astrophysical gases. For oblate modes (short-wavelength incompressive modes elongated in the direction of the mean flow), a second-order ordinary differential equation is derived that can be used to assess the stability of any time-dependent flow with planar, cylindrical or spherical symmetry. Upon further restricting the analysis to homologous flows, it is shown that a monatomic gas is governed by the Schwarzschild criterion for buoyant stability. Under buoyantly unstable conditions, both entropy and vorticity fluctuations experience power-law growth in time, with a growth rate that depends upon mean flow gradients and, in the absence of dissipative effects, is independent of mode number. If the flow accelerates throughout the implosion, oblate modes amplify by a factor (2C)|N0|ti, where C is the convergence ratio of the implosion, N0 is the initial buoyancy frequency and ti is the implosion time scale. If, instead, the implosion consists of a coasting phase followed by stagnation, oblate modes amplify by a factor exp(π|N0|ts), where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. Even under stable conditions, vorticity fluctuations grow due to the conservation of angular momentum as the gas is compressed. For non-monatomic gases, this additional growth due to compression results in weak oscillatory growth under conditions that would otherwise be buoyantly stable; this over-stability is consistent with the conservation of wave action in the fluid frame. The above analytical results are verified by evolving the complete set of linear equations as an initial value problem, and it is demonstrated that oblate modes are the fastest

  10. Buoyancy instability of homologous implosions

    DOE PAGES

    Johnson, B. M.

    2015-06-15

    With this study, I consider the hydrodynamic stability of imploding ideal gases as an idealized model for inertial confinement fusion capsules, sonoluminescent bubbles and the gravitational collapse of astrophysical gases. For oblate modes (short-wavelength incompressive modes elongated in the direction of the mean flow), a second-order ordinary differential equation is derived that can be used to assess the stability of any time-dependent flow with planar, cylindrical or spherical symmetry. Upon further restricting the analysis to homologous flows, it is shown that a monatomic gas is governed by the Schwarzschild criterion for buoyant stability. Under buoyantly unstable conditions, both entropy andmore » vorticity fluctuations experience power-law growth in time, with a growth rate that depends upon mean flow gradients and, in the absence of dissipative effects, is independent of mode number. If the flow accelerates throughout the implosion, oblate modes amplify by a factor (2C)|N0|ti, where C is the convergence ratio of the implosion, N0 is the initial buoyancy frequency and ti is the implosion time scale. If, instead, the implosion consists of a coasting phase followed by stagnation, oblate modes amplify by a factor exp(π|N0|ts), where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. Even under stable conditions, vorticity fluctuations grow due to the conservation of angular momentum as the gas is compressed. For non-monatomic gases, this additional growth due to compression results in weak oscillatory growth under conditions that would otherwise be buoyantly stable; this over-stability is consistent with the conservation of wave action in the fluid frame. The above analytical results are verified by evolving the complete set of linear equations as an initial value problem, and it is demonstrated that oblate modes are the fastest-growing modes and that high mode numbers are required to reach this limit (Legendre mode ℓ ≳ 100

  11. The homologous recombination system of Ustilago maydis

    PubMed Central

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of Ustilago maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins. PMID:18502156

  12. Persistent homology analysis of phase transitions

    NASA Astrophysics Data System (ADS)

    Donato, Irene; Gori, Matteo; Pettini, Marco; Petri, Giovanni; De Nigris, Sarah; Franzosi, Roberto; Vaccarino, Francesco

    2016-05-01

    Persistent homology analysis, a recently developed computational method in algebraic topology, is applied to the study of the phase transitions undergone by the so-called mean-field XY model and by the ϕ4 lattice model, respectively. For both models the relationship between phase transitions and the topological properties of certain submanifolds of configuration space are exactly known. It turns out that these a priori known facts are clearly retrieved by persistent homology analysis of dynamically sampled submanifolds of configuration space.

  13. Dualities in Persistent (Co)Homology

    SciTech Connect

    de Silva, Vin; Morozov, Dmitriy; Vejdemo-Johansson, Mikael

    2011-09-16

    We consider sequences of absolute and relative homology and cohomology groups that arise naturally for a filtered cell complex. We establishalgebraic relationships between their persistence modules, and show that they contain equivalent information. We explain how one can use the existingalgorithm for persistent homology to process any of the four modules, and relate it to a recently introduced persistent cohomology algorithm. Wepresent experimental evidence for the practical efficiency of the latter algorithm.

  14. On the hodological criterion for homology

    PubMed Central

    Faunes, Macarena; Francisco Botelho, João; Ahumada Galleguillos, Patricio; Mpodozis, Jorge

    2015-01-01

    Owen's pre-evolutionary definition of a homolog as “the same organ in different animals under every variety of form and function” and its redefinition after Darwin as “the same trait in different lineages due to common ancestry” entail the same heuristic problem: how to establish “sameness.”Although different criteria for homology often conflict, there is currently a generalized acceptance of gene expression as the best criterion. This gene-centered view of homology results from a reductionist and preformationist concept of living beings. Here, we adopt an alternative organismic-epigenetic viewpoint, and conceive living beings as systems whose identity is given by the dynamic interactions between their components at their multiple levels of composition. We posit that there cannot be an absolute homology criterion, and instead, homology should be inferred from comparisons at the levels and developmental stages where the delimitation of the compared trait lies. In this line, we argue that neural connectivity, i.e., the hodological criterion, should prevail in the determination of homologies between brain supra-cellular structures, such as the vertebrate pallium. PMID:26157357

  15. Structural Studies of the Final Enzyme in the alpha-Aminoadipate Pathway-Saccharopine Dehydrogenase from Saccharomyces cerevisiae

    SciTech Connect

    Burk,D.; Hwang, J.; Kwok, E.; Marrone, L.; Goodfellow, V.; Dmitrienko, G.; Berghuis, A.

    2007-01-01

    The 1.64 Angstroms structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogense, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.

  16. Effects of pentamidine isethionate on Saccharomyces cerevisiae.

    PubMed

    Ludewig, G; Williams, J M; Li, Y; Staben, C

    1994-05-01

    We used Saccharomyces cerevisiae as a model system in which to examine the mechanism of action of the anti-Pneumocystis drug pentamidine. Pentamidine at low concentrations inhibited S. cerevisiae growth on nonfermentable carbon sources (50% inhibitory concentration [IC50] of 1.25 micrograms/ml in glycerol). Pentamidine inhibited growth on fermentable energy sources only at much higher concentrations (IC50 of 250 micrograms/ml in glucose). Inhibition at low pentamidine concentrations in glycerol was due to cytostatic activity rather than cytotoxic or mutagenic activity. Pentamidine also rapidly inhibited respiration by intact yeast cells, although inhibitory concentrations were much higher than those inhibitory to growth (IC50 of 100 micrograms/ml for respiration). Pentamidine also induced petite mutations, although only at concentrations much higher than those required for growth inhibition. These results suggest that a function essential for respiratory growth is inhibited by pentamidine and that pentamidine affects mitochondrial processes. We propose the hypothesis that the primary cellular target of pentamidine in S. cerevisiae is the mitochondrion.

  17. Saccharomyces cerevisiae metabolism in ecological context

    PubMed Central

    Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R.

    2016-01-01

    The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype–phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype–environment–phenotype relationships. PMID:27634775

  18. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  19. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

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

  20. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    PubMed

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

    2015-01-01

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

  1. Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

    PubMed

    Wu, Hong; Ito, Kiyoshi; Shimoi, Hitoshi

    2005-11-01

    Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.

  2. Isolation and characterization of WHI3, a size-control gene of Saccharomyces cerevisiae.

    PubMed Central

    Nash, R S; Volpe, T; Futcher, B

    2001-01-01

    WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function. PMID:11290704

  3. Cell death caused by excision of centromeric DNA from a chromosome in Saccharomyces cerevisiae.

    PubMed

    Miyamoto, Akihiro; Yanamoto, Toshiaki; Matsumoto, Takehiro; Hatano, Takushi; Matsuzaki, Hiroaki

    2013-01-01

    If genetically modified organisms (GMOs) are spread through the natural environment, it might affect the natural environment. To help prevent the spread of GMOs, we examined whether it is possible to introduce conditional lethality by excising centromeric DNA from a chromosome by site-specific recombination in Saccharomyces cerevisiae as model organism. First, we constructed haploid cells in which excision of the centromeric DNA from chromosome IV can occur due to recombinase induced by galactose. By this excision, cell death can occur. In diploid cells, cell death can also occur by excision from both homologous chromosomes IV. Furthermore, cell death can occur in the case of chromosome V. A small number of surviving cells appeared with excision of centromeric DNA, and the diploid showed greater viability than the haploid in both chromosomes IV and V. The surviving cells appeared mainly due to deletion of a recombination target site (RS) from the chromosome. PMID:24018677

  4. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

  5. SPO73 and SPO71 Function Cooperatively in Prospore Membrane Elongation During Sporulation in Saccharomyces cerevisiae.

    PubMed

    Parodi, Emily M; Roesner, Joseph M; Huang, Linda S

    2015-01-01

    In the yeast Saccharomyces cerevisiae, cells undergoing sporulation form prospore membranes to surround their meiotic nuclei. The prospore membranes ultimately become the plasma membranes of the new cells. The putative phospholipase Spo1 and the tandem Pleckstrin Homology domain protein Spo71 have previously been shown to be required for prospore membrane development, along with the constitutively expressed Vps13 involved in vacuolar sorting. Here, we utilize genetic analysis, and find that SPO73 is required for proper prospore membrane shape and, like SPO71, is necessary for prospore membrane elongation. Additionally, similar to SPO71, loss of SPO73 partially suppresses spo1Δ. Spo73 localizes to prospore membranes and complexes with Spo71. We also find that phosphatidylserine localizes to the prospore membrane. Our results suggest a model where SPO71 and SPO73 act in opposition to SPO1 to form and elongate prospore membranes, while VPS13 plays a distinct role in prospore membrane development.

  6. Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Alani, E.; Reenan, RAG.; Kolodner, R. D.

    1994-01-01

    The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. PMID:8056309

  7. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

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

  8. Topological and mutational analysis of Saccharomyces cerevisiae Fks1.

    PubMed

    Johnson, Michael E; Edlind, Thomas D

    2012-07-01

    Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.

  9. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

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

  10. Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

    PubMed Central

    Edlind, Thomas D.

    2012-01-01

    Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2. PMID:22581527

  11. Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH.

    PubMed

    Conroy, J M; Borzelleca, D C; McDonell, L A

    1982-01-01

    Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.

  12. HOVERGEN: a database of homologous vertebrate genes.

    PubMed Central

    Duret, L; Mouchiroud, D; Gouy, M

    1994-01-01

    Comparison of homologous genes is a major step for many studies related to genome structure, function or evolution. Similarity search programs easily find genes homologous to a given sequence. However, only very tedious manual procedures allow the retrieval of all sets of homologous genes sequenced for a given set of species. Moreover, this search often generates errors due to the complexity of data to be managed simultaneously: phylogenetic trees, alignments, taxonomy, sequences and related information. HOVERGEN helps to solve these problems by integrating all this information. HOVERGEN corresponds to GenBank sequences from all vertebrate species, with some data corrected, clarified, or completed, notably to address the problem of redundancy. Coding sequences have been classified in gene families. Protein multiple alignments and phylogenetic trees have been calculated for each family. Sequences and related information have been structured in an ACNUC database which permits complex selections. A graphical interface has been developed to visualize and edit trees. Genes are displayed in color, according to their taxonomy. Users have directly access to all information attached to sequences and to multiple alignments simply by clicking on genes. This graphical tool gives thus a rapid and simple access to all data necessary to interpret homology relationships between genes. HOVERGEN allows the user to easily select sets of homologous vertebrate genes, and thus is particularly useful for comparative sequence analysis, or molecular evolution studies. Images PMID:8036164

  13. Homological scaffolds of brain functional networks.

    PubMed

    Petri, G; Expert, P; Turkheimer, F; Carhart-Harris, R; Nutt, D; Hellyer, P J; Vaccarino, F

    2014-12-01

    Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186-198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects-homological cycles-associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle,we apply these tools to compare resting state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin-the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain's functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo.

  14. Homological scaffolds of brain functional networks

    PubMed Central

    Petri, G.; Expert, P.; Turkheimer, F.; Carhart-Harris, R.; Nutt, D.; Hellyer, P. J.; Vaccarino, F.

    2014-01-01

    Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186–198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects—homological cycles—associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle, we apply these tools to compare resting-state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin—the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain's functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo. PMID:25401177

  15. Homological scaffolds of brain functional networks.

    PubMed

    Petri, G; Expert, P; Turkheimer, F; Carhart-Harris, R; Nutt, D; Hellyer, P J; Vaccarino, F

    2014-12-01

    Networks, as efficient representations of complex systems, have appealed to scientists for a long time and now permeate many areas of science, including neuroimaging (Bullmore and Sporns 2009 Nat. Rev. Neurosci. 10, 186-198. (doi:10.1038/nrn2618)). Traditionally, the structure of complex networks has been studied through their statistical properties and metrics concerned with node and link properties, e.g. degree-distribution, node centrality and modularity. Here, we study the characteristics of functional brain networks at the mesoscopic level from a novel perspective that highlights the role of inhomogeneities in the fabric of functional connections. This can be done by focusing on the features of a set of topological objects-homological cycles-associated with the weighted functional network. We leverage the detected topological information to define the homological scaffolds, a new set of objects designed to represent compactly the homological features of the correlation network and simultaneously make their homological properties amenable to networks theoretical methods. As a proof of principle,we apply these tools to compare resting state functional brain activity in 15 healthy volunteers after intravenous infusion of placebo and psilocybin-the main psychoactive component of magic mushrooms. The results show that the homological structure of the brain's functional patterns undergoes a dramatic change post-psilocybin, characterized by the appearance of many transient structures of low stability and of a small number of persistent ones that are not observed in the case of placebo. PMID:25401177

  16. Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae.

    PubMed

    Skaar, Karin; Korza, Henryk J; Tarry, Michael; Sekyrova, Petra; Högbom, Martin

    2015-01-01

    Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies. PMID:26218426

  17. Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

    PubMed Central

    Lefrançois, Philippe; Rockmill, Beth; Xie, Pingxing; Roeder, G. Shirleen; Snyder, Michael

    2016-01-01

    During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition. PMID:27768699

  18. Irradiated homologous costal cartilage for augmentation rhinoplasty

    SciTech Connect

    Lefkovits, G. )

    1990-10-01

    Although the ideal reconstructive material for augmentation rhinoplasty continues to challenge plastic surgeons, there exists no report in the literature that confines the use of irradiated homologous costal cartilage, first reported by Dingman and Grabb in 1961, to dorsal nasal augmentation. The purpose of this paper is to present a retrospective analysis of the author's experience using irradiated homologous costal cartilage in augmentation rhinoplasty. Twenty-seven dorsal nasal augmentations were performed in 24 patients between 16 and 49 years of age with a follow-up ranging from 1 to 27 months. Good-to-excellent results were achieved in 83.3% (20 of 24). Poor results requiring revision were found in 16.7% (4 of 24). Complication rates included 7.4% infection (2 of 27) and 14.8% warping (4 of 27). The resorption rate was zero. These results compare favorably with other forms of nasal augmentation. Advantages and disadvantages of irradiated homologous costal cartilage are discussed.

  19. Coronal Magnetic Structures for Homologous Eruptions

    NASA Astrophysics Data System (ADS)

    Lee, J.; Liu, C.; Jing, J.; Chae, J.

    2015-12-01

    Many studies have been made on homologous eruptions for their importance in understanding the flare energy build-up and release processes. We study the homologous eruptions that occurred in three active regions, NOAA 11444, 11283, and 12192, with emphasis on the coronal quantities derived from the nonlinear force-free field (NLFFF) extrapolation. The quantities include magnetic energy, electric current, and magnetic twist number, and decay index, computed from the high cadence photospheric vector magnetograms of the Helioseismic and Magnetic Imager (HMI) on board the Solar Dynamic Observatory (SDO). In addition, photospheric magnetic flux, flare ribbons and overlying field distribution are also examined to determine the changes associated with each eruption. As main results, we will present the difference between the homology of confined eruptions and that of eruptive ones, and variations of the coronal quantities with flare strength.

  20. Solar core homology, solar neutrinos and helioseismology

    SciTech Connect

    Bludman, S.A.; Kennedy, D.C.

    1995-12-31

    Precise numerical standard solar models (SSMs) now agree with one another and with helioseismological observations in the convective and outer radiative zones. Nevertheless these models obscure how luminosity, neutrino production and g-mode core helioseismology depend on such inputs as opacity and nuclear cross sections. Although the Sun is not homologous, its inner core by itself is chemically evolved and almost homologous, because of its compactness, radiative energy transport, and ppI-dominated luminosity production. We apply luminosity-fixed homology transformations to the core to estimate theoretical uncertainties in the SSM and to obtain a broad class of non-SSMs, parameterized by central temperature and density and purely radiative energy transport in the core. 25 refs., 3 figs., 3 tabs.

  1. Cloning and characterisation of the S. pombe rad15 gene, a homologue to the S. cerevisiae RAD3 and human ERCC2 genes.

    PubMed Central

    Murray, J M; Doe, C L; Schenk, P; Carr, A M; Lehmann, A R; Watts, F Z

    1992-01-01

    The RAD3 gene of Saccharomyces cerevisiae encodes an ATP-dependent 5'-3' DNA helicase, which is involved in excision repair of ultraviolet radiation damage. By hybridisation of a Schizosaccharomyces pombe genomic library with a RAD3 gene probe we have isolated the S. pombe homologue of RAD3. We have also cloned the rad15 gene of S. pombe by complementation of radiation-sensitive phenotype of the rad15 mutant. Comparison of the restriction map and DNA sequence, shows that the S. pombe rad15 gene is identical to the gene homologous to S. cerevisiae RAD3, identified by hybridisation. The S. pombe rad15.P mutant is highly sensitive to UV radiation, but only slightly sensitive to ionising radiation, as expected for a mutant defective in excision repair. DNA sequence analysis of the rad15 gene indicates an open reading frame of 772 amino acids, and this is consistent with a transcript size of 2.6 kb as detected by Northern analysis. The predicted rad15 protein has 65% identity to RAD3 and 55% identity to the human homologue ERCC2. This homology is particularly striking in the regions identified as being conserved in a group of DNA helicases. Gene deletion experiments indicate that, like the S. cerevisiae RAD3 gene, the S. pombe rad15 gene is essential for viability, suggesting that the protein product has a role in cell proliferation and not solely in DNA repair. Images PMID:1319571

  2. Crystal structure of an archaeal actin homolog.

    PubMed

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  3. Saccharomyces cerevisiae CWH43 Is Involved in the Remodeling of the Lipid Moiety of GPI Anchors to Ceramides

    PubMed Central

    Umemura, Mariko; Fujita, Morihisa; Yoko-o, Takehiko; Fukamizu, Akiyoshi

    2007-01-01

    The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities. PMID:17761529

  4. Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

    PubMed

    Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

    2016-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated. PMID:27687229

  5. Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

    PubMed

    Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

    2016-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

  6. Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae

    PubMed Central

    Konte, Tilen; Terpitz, Ulrich; Plemenitaš, Ana

    2016-01-01

    The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus. PMID:27379041

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

    PubMed

    Liu, Jidong; Zhou, Jingwen; Chen, Jian

    2013-06-01

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

  8. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  9. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  10. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed Central

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-01

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. PMID:15065659

  11. Ycs4 is Required for Efficient Double-Strand Break Formation and Homologous Recombination During Meiosis.

    PubMed

    Hong, Soogil; Choi, Eui-Hwan; Kim, Keun Pil

    2015-07-01

    Condensin is not only responsible for chromosome condensation, but is also involved in double-strand break (DSB) processing in the cell cycle. During meiosis, the condensin complex serves as a component of the meiotic chromosome axis, and mediates both proper assembly of the synaptonemal complex and DSB repair, in order to ensure proper homologous chromosome segregation. Here, we used the budding yeast Saccharomyces cerevisiae to show that condensin participates in a variety of chromosome organization processes and exhibits crucial molecular functions that contribute to meiotic recombination during meiotic prophase I. We demonstrate that Ycs4 is required for efficient DSB formation and establishing homolog bias at the early stage of meiotic prophase I, which allows efficient formation of interhomolog recombination products. In the Ycs4 meiosis-specific allele (ycs4S), interhomolog products were formed at substantial levels, but with the same reduction in crossovers and noncrossovers. We further show that, in prophase chromosomal events, ycs4S relieved the defects in the progression of recombination interactions induced as a result of the absence of Rec8. These results suggest that condensin is a crucial coordinator of the recombination process and chromosome organization during meiosis.

  12. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Eldarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  13. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  14. An Arabidopsis Homolog of Yeast ATG6/VPS30 Is Essential for Pollen Germination1[W

    PubMed Central

    Fujiki, Yuki; Yoshimoto, Kohki; Ohsumi, Yoshinori

    2007-01-01

    Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination. PMID:17259285

  15. What Evidence Is There for the Homology of Protein-Protein Interactions?

    PubMed Central

    Lewis, Anna C. F.; Jones, Nick S.; Porter, Mason A.; Deane, Charlotte M.

    2012-01-01

    The notion that sequence homology implies functional similarity underlies much of computational biology. In the case of protein-protein interactions, an interaction can be inferred between two proteins on the basis that sequence-similar proteins have been observed to interact. The use of transferred interactions is common, but the legitimacy of such inferred interactions is not clear. Here we investigate transferred interactions and whether data incompleteness explains the lack of evidence found for them. Using definitions of homology associated with functional annotation transfer, we estimate that conservation rates of interactions are low even after taking interactome incompleteness into account. For example, at a blastp -value threshold of , we estimate the conservation rate to be about between S. cerevisiae and H. sapiens. Our method also produces estimates of interactome sizes (which are similar to those previously proposed). Using our estimates of interaction conservation we estimate the rate at which protein-protein interactions are lost across species. To our knowledge, this is the first such study based on large-scale data. Previous work has suggested that interactions transferred within species are more reliable than interactions transferred across species. By controlling for factors that are specific to within-species interaction prediction, we propose that the transfer of interactions within species might be less reliable than transfers between species. Protein-protein interactions appear to be very rarely conserved unless very high sequence similarity is observed. Consequently, inferred interactions should be used with care. PMID:23028270

  16. Connectivity Homology Enables Inter-Species Network Models of Synthetic Lethality

    PubMed Central

    Jacunski, Alexandra; Dixon, Scott J.; Tatonetti, Nicholas P.

    2015-01-01

    Synthetic lethality is a genetic interaction wherein two otherwise nonessential genes cause cellular inviability when knocked out simultaneously. Drugs can mimic genetic knock-out effects; therefore, our understanding of promiscuous drugs, polypharmacology-related adverse drug reactions, and multi-drug therapies, especially cancer combination therapy, may be informed by a deeper understanding of synthetic lethality. However, the colossal experimental burden in humans necessitates in silico methods to guide the identification of synthetic lethal pairs. Here, we present SINaTRA (Species-INdependent TRAnslation), a network-based methodology that discovers genome-wide synthetic lethality in translation between species. SINaTRA uses connectivity homology, defined as biological connectivity patterns that persist across species, to identify synthetic lethal pairs. Importantly, our approach does not rely on genetic homology or structural and functional similarity, and it significantly outperforms models utilizing these data. We validate SINaTRA by predicting synthetic lethality in S. pombe using S. cerevisiae data, then identify over one million putative human synthetic lethal pairs to guide experimental approaches. We highlight the translational applications of our algorithm for drug discovery by identifying clusters of genes significantly enriched for single- and multi-drug cancer therapies. PMID:26451775

  17. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-29

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

  18. Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

    PubMed Central

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence. PMID:24614282

  19. Assessing chronological aging in Saccharomyces cerevisiae.

    PubMed

    Hu, Jia; Wei, Min; Mirisola, Mario G; Longo, Valter D

    2013-01-01

    Saccharomyces cerevisiae is one of the most studied model organisms for the identification of genes and mechanisms that affect aging. The chronological lifespan (CLS) assay, which monitors the survival of a non-dividing population, is one of the two methods to study aging in yeast. To eliminate potential artifacts and identify genes and signaling pathways that may also affect aging in higher eukaryotes, it is important to determine CLS by multiple methods. Here, we describe these methods as well as the assays to study macromolecular damage during aging in yeast, with a focus on genomic instability.

  20. Components of microtubular structures in Saccharomyces cerevisiae.

    PubMed Central

    Pillus, L; Solomon, F

    1986-01-01

    Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures. In many of the classical cases, however, the nature of the system chosen has precluded a concurrent genetic analysis. The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics. We show here that this microtubule structure also can be examined biochemically. The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells. In this way, microtubule-associated proteins of the yeast spindle are identified. Images PMID:3517870

  1. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

    PubMed Central

    Carr, A M; Sheldrick, K S; Murray, J M; al-Harithy, R; Watts, F Z; Lehmann, A R

    1993-01-01

    Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2. Images PMID:8464724

  2. Coupling Unbiased Mutagenesis to High-throughput DNA Sequencing Uncovers Functional Domains in the Ndc80 Kinetochore Protein of Saccharomyces cerevisiae

    PubMed Central

    Tien, Jerry F.; Fong, Kimberly K.; Umbreit, Neil T.; Payen, Celia; Zelter, Alex; Asbury, Charles L.; Dunham, Maitreya J.; Davis, Trisha N.

    2013-01-01

    During mitosis, kinetochores physically link chromosomes to the dynamic ends of spindle microtubules. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain. Ndc80 is also required for recruiting other components to the kinetochore and responding to mitotic regulatory signals. While the calponin homology domain has been the focus of biochemical and structural characterization, the function of the remainder of Ndc80 is poorly understood. Here, we utilized a new approach that couples high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae. We identified domains in previously uncharacterized regions of Ndc80 that are essential for its function in vivo. We show that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex. Furthermore, a mutation in this hairpin abolishes the ability of the Dam1 complex to strengthen microtubule attachments made by the Ndc80 complex. Finally, we defined a C-terminal segment of Ndc80 required for tetramerization of the Ndc80 complex in vivo. This unbiased mutagenesis approach can be generally applied to genes in S. cerevisiae to identify functional properties and domains. PMID:23833183

  3. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics.

    PubMed

    Suhandynata, Raymond T; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.

  4. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

    PubMed Central

    Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  5. Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.

    PubMed Central

    Fiorentini, P; Huang, K N; Tishkoff, D X; Kolodner, R D; Symington, L S

    1997-01-01

    We previously described a 5'-3' exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5'-3' exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair. PMID:9111347

  6. Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae.

    PubMed

    Fasullo, M; Dave, P

    1994-04-01

    Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cerevisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MATlocus. The difference in levels of stimulation between MATa/MAT alpha diploid and MAT alpha haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m2), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MAT alpha gene was introduced by DNA transformation into a MATa/mat alpha::LEU2+ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATa gene was introduced by DNA transformation into a MAT alpha haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV. The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.

  7. Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

    PubMed

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

    2016-06-20

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

  8. [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.

  9. Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

    PubMed

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

    2016-06-20

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

  10. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

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

  11. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

    PubMed

    Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

    2000-04-01

    Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into

  12. Redesigning Aldolase Stereoselectivity by Homologous Grafting.

    PubMed

    Bisterfeld, Carolin; Classen, Thomas; Küberl, Irene; Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity. PMID:27327271

  13. Homologous Pairing between Long DNA Double Helices

    NASA Astrophysics Data System (ADS)

    Mazur, Alexey K.

    2016-04-01

    Molecular recognition between two double stranded (ds) DNA with homologous sequences may not seem compatible with the B-DNA structure because the sequence information is hidden when it is used for joining the two strands. Nevertheless, it has to be invoked to account for various biological data. Using quantum chemistry, molecular mechanics, and hints from recent genetics experiments, I show here that direct recognition between homologous dsDNA is possible through the formation of short quadruplexes due to direct complementary hydrogen bonding of major-groove surfaces in parallel alignment. The constraints imposed by the predicted structures of the recognition units determine the mechanism of complexation between long dsDNA. This mechanism and concomitant predictions agree with the available experimental data and shed light upon the sequence effects and the possible involvement of topoisomerase II in the recognition.

  14. Redesigning Aldolase Stereoselectivity by Homologous Grafting

    PubMed Central

    Henßen, Birgit; Metz, Alexander; Gohlke, Holger; Pietruszka, Jörg

    2016-01-01

    The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity. PMID:27327271

  15. Khovanov homology of graph-links

    SciTech Connect

    Nikonov, Igor M

    2012-08-31

    Graph-links arise as the intersection graphs of turning chord diagrams of links. Speaking informally, graph-links provide a combinatorial description of links up to mutations. Many link invariants can be reformulated in the language of graph-links. Khovanov homology, a well-known and useful knot invariant, is defined for graph-links in this paper (in the case of the ground field of characteristic two). Bibliography: 14 titles.

  16. Homology and phylogeny and their automated inference

    NASA Astrophysics Data System (ADS)

    Fuellen, Georg

    2008-06-01

    The analysis of the ever-increasing amount of biological and biomedical data can be pushed forward by comparing the data within and among species. For example, an integrative analysis of data from the genome sequencing projects for various species traces the evolution of the genomes and identifies conserved and innovative parts. Here, I review the foundations and advantages of this “historical” approach and evaluate recent attempts at automating such analyses. Biological data is comparable if a common origin exists (homology), as is the case for members of a gene family originating via duplication of an ancestral gene. If the family has relatives in other species, we can assume that the ancestral gene was present in the ancestral species from which all the other species evolved. In particular, describing the relationships among the duplicated biological sequences found in the various species is often possible by a phylogeny, which is more informative than homology statements. Detecting and elaborating on common origins may answer how certain biological sequences developed, and predict what sequences are in a particular species and what their function is. Such knowledge transfer from sequences in one species to the homologous sequences of the other is based on the principle of ‘my closest relative looks and behaves like I do’, often referred to as ‘guilt by association’. To enable knowledge transfer on a large scale, several automated ‘phylogenomics pipelines’ have been developed in recent years, and seven of these will be described and compared. Overall, the examples in this review demonstrate that homology and phylogeny analyses, done on a large (and automated) scale, can give insights into function in biology and biomedicine.

  17. The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae

    PubMed Central

    1993-01-01

    Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305- 1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth. PMID:8276900

  18. Advances in Homology Protein Structure Modeling

    PubMed Central

    Xiang, Zhexin

    2007-01-01

    Homology modeling plays a central role in determining protein structure in the structural genomics project. The importance of homology modeling has been steadily increasing because of the large gap that exists between the overwhelming number of available protein sequences and experimentally solved protein structures, and also, more importantly, because of the increasing reliability and accuracy of the method. In fact, a protein sequence with over 30% identity to a known structure can often be predicted with an accuracy equivalent to a low-resolution X-ray structure. The recent advances in homology modeling, especially in detecting distant homologues, aligning sequences with template structures, modeling of loops and side chains, as well as detecting errors in a model, have contributed to reliable prediction of protein structure, which was not possible even several years ago. The ongoing efforts in solving protein structures, which can be time-consuming and often difficult, will continue to spur the development of a host of new computational methods that can fill in the gap and further contribute to understanding the relationship between protein structure and function. PMID:16787261

  19. COMPASS server for remote homology inference.

    PubMed

    Sadreyev, Ruslan I; Tang, Ming; Kim, Bong-Hyun; Grishin, Nick V

    2007-07-01

    COMPASS is a method for homology detection and local alignment construction based on the comparison of multiple sequence alignments (MSAs). The method derives numerical profiles from given MSAs, constructs local profile-profile alignments and analytically estimates E-values for the detected similarities. Until now, COMPASS was only available for download and local installation. Here, we present a new web server featuring the latest version of COMPASS, which provides (i) increased sensitivity and selectivity of homology detection; (ii) longer, more complete alignments; and (iii) faster computational speed. After submission of the query MSA or single sequence, the server performs searches versus a user-specified database. The server includes detailed and intuitive control of the search parameters. A flexible output format, structured similarly to BLAST and PSI-BLAST, provides an easy way to read and analyze the detected profile similarities. Brief help sections are available for all input parameters and output options, along with detailed documentation. To illustrate the value of this tool for protein structure-functional prediction, we present two examples of detecting distant homologs for uncharacterized protein families. Available at http://prodata.swmed.edu/compass. PMID:17517780

  20. Dental homologies in lamniform sharks (Chondrichthyes: Elasmobranchii).

    PubMed

    Shimada, Kenshu

    2002-01-01

    The dentitions of lamniform sharks are said to exhibit a unique heterodonty called the "lamnoid tooth pattern." The presence of an inflated hollow "dental bulla" on each jaw cartilage allows the recognition of homologous teeth across most modern macrophagous lamniforms based on topographic correspondence through the "similarity test." In most macrophagous lamniforms, three tooth rows are supported by the upper dental bulla: two rows of large anterior teeth followed by a row of small intermediate teeth. The lower tooth row occluding between the two rows of upper anterior teeth is the first lower anterior tooth row. Like the first and second lower anterior tooth rows, the third lower tooth row is supported by the dental bulla and may be called the first lower intermediate tooth row. The lower intermediate tooth row occludes between the first and second upper lateral tooth rows situated distal to the upper dental bulla, and the rest of the upper and lower tooth rows, all called lateral tooth rows, occlude alternately. Tooth symmetry cannot be used to identify their dental homology. The presence of dental bullae can be regarded as a synapomorphy of Lamniformes and this character is more definable than the "lamnoid tooth pattern." The formation of the tooth pattern appears to be related to the evolution of dental bullae. This study constitutes the first demonstration of supraspecific tooth-to-tooth dental homologies in nonmammalian vertebrates.

  1. Weak homological dimensions and biflat Koethe algebras

    SciTech Connect

    Pirkovskii, A Yu

    2008-06-30

    The homological properties of metrizable Koethe algebras {lambda}(P) are studied. A criterion for an algebra A={lambda}(P) to be biflat in terms of the Koethe set P is obtained, which implies, in particular, that for such algebras the properties of being biprojective, biflat, and flat on the left are equivalent to the surjectivity of the multiplication operator A otimes-hat A{yields}A. The weak homological dimensions (the weak global dimension w.dg and the weak bidimension w.db) of biflat Koethe algebras are calculated. Namely, it is shown that the conditions w.db {lambda}(P)<=1 and w.dg {lambda}(P)<=1 are equivalent to the nuclearity of {lambda}(P); and if {lambda}(P) is non-nuclear, then w.dg {lambda}(P)=w.db {lambda}(P)=2. It is established that the nuclearity of a biflat Koethe algebra {lambda}(P), under certain additional conditions on the Koethe set P, implies the stronger estimate db {lambda}(P), where db is the (projective) bidimension. On the other hand, an example is constructed of a nuclear biflat Koethe algebra {lambda}(P) such that db {lambda}(P)=2 (while w.db {lambda}(P)=1). Finally, it is shown that many biflat Koethe algebras, while not being amenable, have trivial Hochschild homology groups in positive degrees (with arbitrary coefficients). Bibliography: 37 titles.

  2. Regulation of DNA Pairing in Homologous Recombination

    PubMed Central

    Daley, James M.; Gaines, William A.; Kwon, YoungHo; Sung, Patrick

    2014-01-01

    Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3′ ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3′ end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess. PMID:25190078

  3. Homologous recombination in rat germline stem cells.

    PubMed

    Kanatsu-Shinohara, Mito; Kato-Itoh, Megumi; Ikawa, Masahito; Takehashi, Masanori; Sanbo, Makoto; Morioka, Yuka; Tanaka, Takashi; Morimoto, Hiroko; Hirabayashi, Masumi; Shinohara, Takashi

    2011-07-01

    Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.

  4. In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains.

    PubMed

    van der Aa Kühle, Alis; Skovgaard, Kerstin; Jespersen, Lene

    2005-05-01

    The probiotic potential of 18 Saccharomyces cerevisiae strains used for production of foods or beverages or isolated from such, and eight strains of Saccharomyces cerevisiae var. boulardii, was investigated. All strains included were able to withstand pH 2.5 and 0.3% Oxgall. Adhesion to the nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of 3H-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13.6-16.8%) recorded for two isolates from blue veined cheeses. Merely 25% of the S. cerevisiae var. boulardii strains displayed good adhesive properties (16.2-28.0%). The expression of the proinflammatory cytokine IL-1alpha decreased strikingly in IPEC-J2 cells exposed to a Shiga-like toxin 2e producing Escherichia coli strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness. PMID:15878404

  5. Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae.

    PubMed

    Halbrook, J; McEntee, K

    1989-12-15

    An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.

  6. MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

    PubMed Central

    1996-01-01

    The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP- Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion- disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules. PMID:8947554

  7. The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae.

    PubMed

    Rubtsov, P M; Musakhanov, M M; Zakharyev, V M; Krayev, A S; Skryabin, K G; Bayev, A A

    1980-12-11

    The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.

  8. Roles for sphingolipids in Saccharomyces cerevisiae.

    PubMed

    Dickson, Robert C

    2010-01-01

    Studies using Saccharomyces cerevisiae, the common baker's or brewer's yeast, have progressed over the past twenty years from knowing which sphingolipids are present in cells and a basic outline of how they are made to a complete or nearly complete directory of the genes that catalyze their anabolism and catabolism. In addition, cellular processes that depend upon sphingolipids have been identified including protein trafficking/exocytosis, endocytosis and actin cytoskeleton dynamics, membrane microdomains, calcium signaling, regulation of transcription and translation, cell cycle control, stress resistance, nutrient uptake and aging. These will be summarized here along with new data not previously reviewed. Advances in our knowledge of sphingolipids and their roles in yeast are impressive but molecular mechanisms remain elusive and are a primary challenge for further progress in understanding the specific functions of sphingolipids. PMID:20919657

  9. Transcriptional Regulatory Networks in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

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

    2002-10-01

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

  10. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    PubMed

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

    2016-01-01

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

  11. Vestiges of Ent3p/Ent5p function in the giardial epsin homolog.

    PubMed

    Feliziani, Constanza; Valdez Taubas, Javier; Moyano, Sofía; Quassollo, Gonzalo; Poprawski, Joanna E; Wendland, Beverly; Touz, Maria C

    2016-04-01

    An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae. This protein, named GlENTHp (for G. lamblia ENTH protein), is involved in Giardia in endocytosis and in protein trafficking from the ER to the vacuoles, fulfilling the function of the ENTH proteins epsin and epsinR, respectively. There are two orthologs of epsin, Ent1p and Ent2p, and two orthologs of epsinR, Ent3p and Ent5p in S. cerevisiae. Although the expression of GlENTHp neither complemented growth in the ent1Δent2Δ mutant nor restored the GFP-Cps1 vacuolar trafficking defect in ent3Δent5Δ, it interfered with the normal function of Ent3/5 in the wild-type strain. The phenotype observed is linked to a defect in Cps1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as dominant negative epsinR in yeast cells reinforces the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to their evolution into different taxa.

  12. Vestiges of Ent3p/Ent5p function in the giardial epsin homolog.

    PubMed

    Feliziani, Constanza; Valdez Taubas, Javier; Moyano, Sofía; Quassollo, Gonzalo; Poprawski, Joanna E; Wendland, Beverly; Touz, Maria C

    2016-04-01

    An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae. This protein, named GlENTHp (for G. lamblia ENTH protein), is involved in Giardia in endocytosis and in protein trafficking from the ER to the vacuoles, fulfilling the function of the ENTH proteins epsin and epsinR, respectively. There are two orthologs of epsin, Ent1p and Ent2p, and two orthologs of epsinR, Ent3p and Ent5p in S. cerevisiae. Although the expression of GlENTHp neither complemented growth in the ent1Δent2Δ mutant nor restored the GFP-Cps1 vacuolar trafficking defect in ent3Δent5Δ, it interfered with the normal function of Ent3/5 in the wild-type strain. The phenotype observed is linked to a defect in Cps1 localization and α-factor mating pheromone maturation. The finding that GlENTHp acts as dominant negative epsinR in yeast cells reinforces the phylogenetic data showing that GlENTHp belongs to the epsinR subfamily present in eukaryotes prior to their evolution into different taxa. PMID:26851076

  13. RAD59 is Required for Efficient Repair of Simultaneous Double-Strand Breaks Resulting in Translocations in Saccharomyces cerevisiae

    PubMed Central

    Pannunzio, Nicholas R.; Manthey, Glenn M.; Bailis, Adam M.

    2008-01-01

    Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly-efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300 bp and 60 bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure. PMID:18373960

  14. Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases.

    PubMed Central

    Martin, G; Keller, W

    1996-01-01

    We have tested deletion and substitution mutants of bovine poly(A) polymerase, and have identified a small region that overlaps with a nuclear localization signal and binds to the RNA primer. Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis. Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases. This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor. Images PMID:8665867

  15. Recovery of Saccharomyces cerevisiae from ethanol - induced growth inhibition

    SciTech Connect

    Walker-Caprioglio, H.M.; Rodriguez, R.J.; Parks, L.W.

    1985-09-01

    Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the inoculum.

  16. Railway vehicle performance optimisation using virtual homologation

    NASA Astrophysics Data System (ADS)

    Magalhães, H.; Madeira, J. F. A.; Ambrósio, J.; Pombo, J.

    2016-09-01

    Unlike regular automotive vehicles, which are designed to travel in different types of roads, railway vehicles travel mostly in the same route during their life cycle. To accept the operation of a railway vehicle in a particular network, a homologation process is required according to local standard regulations. In Europe, the standards EN 14363 and UIC 518, which are used for railway vehicle acceptance, require on-track tests and/or numerical simulations. An important advantage of using virtual homologation is the reduction of the high costs associated with on-track tests by studying the railway vehicle performance in different operation conditions. This work proposes a methodology for the improvement of railway vehicle design with the objective of its operation in selected railway tracks by using optimisation. The analyses required for the vehicle improvement are performed under control of the optimisation method global and local optimisation using direct search. To quantify the performance of the vehicle, a new objective function is proposed, which includes: a Dynamic Performance Index, defined as a weighted sum of the indices obtained from the virtual homologation process; the non-compensated acceleration, which is related to the operational velocity; and a penalty associated with cases where the vehicle presents an unacceptable dynamic behaviour according to the standards. Thus, the optimisation process intends not only to improve the quality of the vehicle in terms of running safety and ride quality, but also to increase the vehicle availability via the reduction of the time for a journey while ensuring its operational acceptance under the standards. The design variables include the suspension characteristics and the operational velocity of the vehicle, which are allowed to vary in an acceptable range of variation. The results of the optimisation lead to a global minimum of the objective function in which the suspensions characteristics of the vehicle are

  17. Purification, functional reconstitution, and characterization of the Saccharomyces cerevisiae isoprenylcysteine carboxylmethyltransferase Ste14p.

    PubMed

    Anderson, Jessica L; Frase, Hilary; Michaelis, Susan; Hrycyna, Christine A

    2005-02-25

    Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and alpha-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-beta-d-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure.

  18. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    PubMed

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2016-03-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  19. Cloning of a Cryptococcus neoformans gene, GPA1, encoding a G-protein alpha-subunit homolog.

    PubMed Central

    Tolkacheva, T; McNamara, P; Piekarz, E; Courchesne, W

    1994-01-01

    We have isolated a gene, GPA1, from Cryptococcus neoformans by the PCR technique. DNA sequencing of the GPA1 clone suggested that it encodes a protein homologous to the G-protein alpha-subunit family. Comparison of the deduced amino acid sequence of the GPA1-encoded protein revealed that it is about 45% identical to several mammalian Gi alpha subunits and 48% identical to the G alpha protein Gpa2 from Saccharomyces cerevisiae. G alpha proteins are known to be involved in mating of other yeasts, such as S. cerevisiae and Schizosaccharomyces pombe. Southern analysis demonstrated that GPA1 is present in a single copy within the Cryptococcus genome. Isolation of the cDNA for GPA1 confirmed that the gene contains six introns within the coding region. The GPA1 transcript was identified by Northern (RNA) analysis as a 1.6-kb RNA present in exponentially growing cells of both the alpha and a mating types. Moreover, the abundance of this transcript increased in cells shifted to starvation medium. Coincubation of alpha and a cells on starvation medium is required for mating of cryptococcal cells. Thus, our results are consistent with the involvement of C. neoformans GPA1 in mating. Images PMID:8005675

  20. The antifungal properties of a 2S albumin-homologous protein from passion fruit seeds involve plasma membrane permeabilization and ultrastructural alterations in yeast cells.

    PubMed

    Agizzio, Ana Paula; Da Cunha, Maura; Carvalho, André O; Oliveira, Marco Antônio; Ribeiro, Suzanna F F; Gomes, Valdirene M

    2006-10-01

    Different types of antimicrobial proteins were purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins and 2S albumins. It has become clear that these groups of proteins play an important role in the protection of plants from microbial infection. Recent results from our laboratory have shown that the defense-related proteins from passion fruit seeds, named Pf1 and Pf2 (which show sequence homology with 2S albumins), inhibit fungal growth and glucose-stimulated acidification of the medium by Saccharomyces cerevisiae cells. The aim of this study was to determine whether 2S albumins from passion fruit seeds induce plasma membrane permeabilization and cause morphological alterations in yeast cells. Initially, we used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in S. cerevisiae cells. When viewed with a confocal laser microscope, S. cervisiae cells showed strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. 2S albumins also inhibited glucose-stimulated acidification of the medium by S. cerevisiae cells, which indicates a probable impairment of fungal metabolism. The microscopical analysis of the yeast cells treated with 2S albumins demonstrated several morphological alterations in cell shape, cell surface, cell wall and bud formation, as well as in the organization of intracellular organelles. PMID:25193649

  1. SANSparallel: interactive homology search against Uniprot.

    PubMed

    Somervuo, Panu; Holm, Liisa

    2015-07-01

    Proteins evolve by mutations and natural selection. The network of sequence similarities is a rich source for mining homologous relationships that inform on protein structure and function. There are many servers available to browse the network of homology relationships but one has to wait up to a minute for results. The SANSparallel webserver provides protein sequence database searches with immediate response and professional alignment visualization by third-party software. The output is a list, pairwise alignment or stacked alignment of sequence-similar proteins from Uniprot, UniRef90/50, Swissprot or Protein Data Bank. The stacked alignments are viewed in Jalview or as sequence logos. The database search uses the suffix array neighborhood search (SANS) method, which has been re-implemented as a client-server, improved and parallelized. The method is extremely fast and as sensitive as BLAST above 50% sequence identity. Benchmarks show that the method is highly competitive compared to previously published fast database search programs: UBLAST, DIAMOND, LAST, LAMBDA, RAPSEARCH2 and BLAT. The web server can be accessed interactively or programmatically at http://ekhidna2.biocenter.helsinki.fi/cgi-bin/sans/sans.cgi. It can be used to make protein functional annotation pipelines more efficient, and it is useful in interactive exploration of the detailed evidence supporting the annotation of particular proteins of interest. PMID:25855811

  2. Towards Scalable Optimal Sequence Homology Detection

    SciTech Connect

    Daily, Jeffrey A.; Krishnamoorthy, Sriram; Kalyanaraman, Anantharaman

    2012-12-26

    Abstract—The field of bioinformatics and computational biol- ogy is experiencing a data revolution — experimental techniques to procure data have increased in throughput, improved in accuracy and reduced in costs. This has spurred an array of high profile sequencing and data generation projects. While the data repositories represent untapped reservoirs of rich information critical for scientific breakthroughs, the analytical software tools that are needed to analyze large volumes of such sequence data have significantly lagged behind in their capacity to scale. In this paper, we address homology detection, which is a funda- mental problem in large-scale sequence analysis with numerous applications. We present a scalable framework to conduct large- scale optimal homology detection on massively parallel super- computing platforms. Our approach employs distributed memory work stealing to effectively parallelize optimal pairwise alignment computation tasks. Results on 120,000 cores of the Hopper Cray XE6 supercomputer demonstrate strong scaling and up to 2.42 × 107 optimal pairwise sequence alignments computed per second (PSAPS), the highest reported in the literature.

  3. Homology modelling of human P-glycoprotein.

    PubMed

    Domicevica, Laura; Biggin, Philip C

    2015-10-01

    P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach.

  4. Developmental basis of limb homology in lizards.

    PubMed

    Fabrezi, Marissa; Abdala, Virginia; Oliver, María Inés Martínez

    2007-07-01

    Shubin and Alberch (Evol Biol 1986;20:319-387) proposed a scheme of tetrapod limb development based on cartilage morphogenesis that provides the arguments to interpret the homologies of skeletal elements and sets the basis to explain limb specialization through later developmental modification. Morphogenetic evidence emerged from the study of some reptiles, but the availability of data for lizards is limited. Here, the study of adult skeletal variation in 41 lizard taxa and ontogeny in species of Liolaemus and Tupinambis attempts to fill in this gap and provides supporting evidence for the Shubin-Alberch scheme. Six questions are explored. Is there an intermedium in the carpus? Are there two centralia in the carpus? Is there homology among proximal tarsalia of reptiles? Does digit V belong to the digital arch? Is the pisiform an element of the autopodium plan? And should the ossification processes be similar to cartilage morphogenesis? We found the following answers. Some taxa exhibit an ossified element that could represent an intermedium. There is one centrale in the carpus. Development of proximal tarsalia seems to be equivalent with that observed among reptiles. Digit V could arise from the digital arch. Pisiform does not arise as part of the limb plan. And different patterns of ossification occur following a single and conservative cartilaginous configuration. Lizard limb development shows an early pattern common to other reptiles with clear primary axis and digital arch. The pattern then becomes lizard-specific with specialization involving some reduction in prechondrogenic elements. PMID:17415759

  5. Srs2 and Mus81–Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates

    PubMed Central

    Keyamura, Kenji; Arai, Kota

    2016-01-01

    Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81–Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. PMID:27390022

  6. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.

    PubMed Central

    Xiao, W; Derfler, B; Chen, J; Samson, L

    1991-01-01

    We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro. Here we characterize the cloned S. cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage. We isolated a yeast DNA fragment that suppressed alkylation-induced killing and mutation in Escherichia coli ada ogt MTase deficient mutants and produced in these cells a protein similar to the yeast MTase. The cloned yeast fragment was mapped to chromosome IV and DNA sequencing identified an open reading frame, designated MGT1, which encodes a 188 amino acid protein with a molecular weight of 21,500 daltons. An 88 amino acid stretch of the MGT1 protein displays remarkable homology with four bacterial MTases and the human DNA MTase. S.cerevisiae mutants bearing an insertion in the MGT1 gene lacked DNA MTase activity and were very sensitive to alkylation induced killing and mutation. MGT1 transcript levels are not increased in response to DNA alkylation damage, nor is the MGT1 MTase involved in the regulation of the yeast 3-methyladenine DNA glycosylase gene (MAG). Expression of the MGT1 gene in E.coli prevented the induction by alkylating agents of both G:C to A:T and A:T to G:C transition mutations indicating that this eukaryotic MTase repairs both O6MeG and O4MeT in vivo. Images PMID:2065659

  7. Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe

    PubMed Central

    Huh, Dann; Hallacli, Erinc; Lindquist, Susan

    2016-01-01

    Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells. PMID:27736907

  8. Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae.

    PubMed

    Cannon, R D; Jenkinson, H F; Shepherd, M G

    1990-04-01

    An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2-) to Leu+ at a frequency of 2.15 x 10(3) transformants per microgram DNA, and transformed C. albicans SGY-243 (delta ura3) to Ura+ at a frequency of 1.91 x 10(3) transformants per microgram DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5'TTTTATGTTTT3') which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 bp from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2196431

  9. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    PubMed

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  10. A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae.

    PubMed

    Szallies, Alexander; Kubata, Bruno K; Duszenko, Michael

    2002-04-24

    Metacaspases constitute a new group of cysteine proteases homologous to caspases. Heterologous expression of Trypanosoma brucei metacaspase TbMCA4 in the budding yeast Saccharomyces cerevisiae resulted in growth inhibition, mitochondrial dysfunction and clonal death. The metacaspase orthologue of yeast, ScMCA1 (YOR197w), exhibited genetic interaction with WWM1 (YFL010c), which encodes a small WW domain protein. WWM1 overexpression resulted in growth arrest and clonal death, which was suppressed by concomitant overexpression of ScMCA1. GFP-fusion reporters of WWM1, ScMCA1 and TbMCA4 localized to the nucleus. Taken together, we suggest that metacaspases may play a role in nuclear function controlling cellular proliferation coupled to mitochondrial biogenesis.

  11. Force Sensitivity in Saccharomyces cerevisiae Flocculins.

    PubMed

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

    2016-01-01

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

  12. Force Sensitivity in Saccharomyces cerevisiae Flocculins

    PubMed Central

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

    2016-01-01

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

  13. Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

    PubMed

    Alexieva, K I; Klis, F; Wedler, H; Wambutt, R; Venkov, P

    1999-09-01

    Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.

  14. Isolation of three contiguous genes, ACR1, ACR2 and ACR3, involved in resistance to arsenic compounds in the yeast Saccharomyces cerevisiae.

    PubMed

    Bobrowicz, P; Wysocki, R; Owsianik, G; Goffeau, A; Ułaszewski, S

    1997-07-01

    A 4.2 kb region from Saccharomyces cerevisiae chromosome XVI was isolated as a yeast fragment conferring resistance to 7 mM-sodium arsenite (NaAsO2), when put on a multicopy plasmid. Homology searches revealed a cluster of three new open reading frames named ACR1, ACR2 and ACR3. The hypothetical product of the ACR1 gene is similar to the transcriptional regulatory proteins, encoded by YAP1, and YAP2 genes from S. cerevisiae. Disruption of the ACR1 gene conduces to an arsenite and arsenate hypersensitivity phenotype. The ACR2 gene is indispensable for arsenate but not for arsenite resistance. The hypothetical product of the ACR3 gene shows high similarity to the hypothetical membrane protein encoded by Bacillus subtilis ORF1 of the skin element and weak similarity to the ArsB membrane protein of the Staphylococcus aureus arsenical-resistance operon. Overexpression of the ACR3 gene confers an arsenite- but not an arsenate-resistance phenotype. The presence of ACR3 together with ACR2 on a multicopy plasmid expands the resistance phenotype into arsenate. These findings suggest that all three novel genes: ACR1, ACR2 and ACR3 are involved in the arsenical-resistance phenomenon in S. cerevisiae.

  15. Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis.

    PubMed

    Amore, R; Kötter, P; Küster, C; Ciriacy, M; Hollenberg, C P

    1991-12-20

    The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined. In the 5' noncoding region of the P. stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106. The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR). The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae. Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae. None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein. Transformants of S. cerevisiae containing XYL1 of P. stipitis synthesize an active XR. Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S. cerevisiae and Escherichia coli.

  16. Identification of antifungal natural products via Saccharomyces cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and mode of action

    PubMed Central

    Tebbets, Brad; Yu, Zhiguo; Stewart, Douglas; Zhao, Li-Xing; Jiang, Yi; Xu, Li-Hua; Andes, David; Shen, Ben; Klein, Bruce

    2012-01-01

    Since current antifungal drugs have not kept pace with the escalating medical demands of fungal infections, new, effective medications are required. However, antifungal drug discovery is hindered by the evolutionary similarity of mammalian and fungal cells, which results in fungal drug targets having human homologs and drug non-selectivity. The group III hybrid histidine kinases (HHKs) are an attractive drug target since they are conserved in fungi and absent in mammals. We used a Saccharomyces cerevisiae reporter strain that conditionally expresses HHK to establish a high-throughput bioassay to screen microbial extracts natural products for antifungals. We identified macrotetrolides, a group of related ionophores thought to exhibit restricted antifungal activity. In addition to confirming the use of this bioassay for the discovery of antifungal natural products, we demonstrated broader, more potent fungistatic activity of the macrotetrolides against multiple Candida spp., Cryptococcus spp., and Candida albicans in biofilms. Macrotetrolides were also active in an animal model of C. albicans biofilm, but were found to have inconsistent activity against fluconazole-resistant C. albicans, with most isolates resistant to this natural product. The macrotetrolides do not directly target HHKs, but their selective activity against S. cerevisiae grown in galactose (regardless of Drk1 expression) revealed potential new insight into the role of ion transport in the mode of action of these promising antifungal compounds. Thus, this simple, high-throughput bioassay permitted us to screen microbial extracts, identify natural products as antifungal drugs, and expand our understanding of the activity of macrotetrolides. PMID:22928922

  17. Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae.

    PubMed

    Fukunaga, Tomoaki; Cha-Aim, Kamonchai; Hirakawa, Yuki; Sakai, Ryota; Kitagawa, Takao; Nakamura, Mikiko; Nonklang, Sanom; Hoshida, Hisashi; Akada, Rinji

    2013-06-01

    Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre-existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C-rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon.

  18. Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae.

    PubMed

    Kinsella, B T; Larkin, A; Bolton, M; Cantwell, B A

    1991-07-01

    The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed. PMID:1934116

  19. Homologous Recombination—Experimental Systems, Analysis and Significance

    PubMed Central

    Kuzminov, Andrei

    2014-01-01

    Homologous recombination is the most complex of all recombination events that shape genomes and produce material for evolution. Homologous recombination events are exchanges between DNA molecules in the lengthy regions of shared identity, catalyzed by a group of dedicated enzymes. There is a variety of experimental systems in E. coli and Salmonella to detect homologous recombination events of several different kinds. Genetic analysis of homologous recombination reveals three separate phases of this process: pre-synapsis (the early phase), synapsis (homologous strand exchange) and post-synapsis (the late phase). In E. coli, there are at least two independent pathway of the early phase and at least two independent pathways of the late phase. All this complexity is incongruent with the originally ascribed role of homologous recombination as accelerator of genome evolution: there is simply not enough duplication and repetition in enterobacterial genomes for homologous recombination to have a detectable evolutionary role, and therefore not enough selection to maintain such a complexity. At the same time, the mechanisms of homologous recombination are uniquely suited for repair of complex DNA lesions called chromosomal lesions. In fact, the two major classes of chromosomal lesions are recognized and processed by the two individual pathways at the early phase of homologous recombination. It follows, therefore, that homologous recombination events are occasional reflections of the continual recombinational repair, made possible in cases of natural or artificial genome redundancy. PMID:26442506

  20. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

    PubMed

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  1. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity

    PubMed Central

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  2. Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.

    PubMed

    Nagata, S; Tsunetsugu-Yokota, Y; Naito, A; Kaziro, Y

    1983-10-01

    A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu. PMID:6353412

  3. Alignment algorithm for homology modeling and threading.

    PubMed Central

    Alexandrov, N. N.; Luethy, R.

    1998-01-01

    A DNA/protein sequence comparison is a popular computational tool for molecular biologists. Finding a good alignment implies an evolutionary and/or functional relationship between proteins or genomic loci. Sequential similarity between two proteins indicates their structural resemblance, providing a practical approach for structural modeling, when structure of one of these proteins is known. The first step in the homology modeling is a construction of an accurate sequence alignment. The commonly used alignment algorithms do not provide an adequate treatment of the structurally mismatched residues in locally dissimilar regions. We propose a simple modification of the existing alignment algorithm which treats these regions properly and demonstrate how this modification improves sequence alignments in real proteins. PMID:9521100

  4. HOMOLOGOUS CYCLONES IN THE QUIET SUN

    SciTech Connect

    Yu, Xinting; Zhang, Jun; Li, Ting; Zhang, Yuzong; Yang, Shuhong E-mail: zjun@nao.cas.cn E-mail: yuzong@nao.cas.cn

    2014-02-20

    Through observations with the Solar Dynamics Observatory Atmospheric Imaging Assembly (AIA) and Helioseismic and Magnetic Imager, we tracked one rotating network magnetic field (RNF) near the solar equator. It lasted for more than 100 hr, from 2013 February 23 to 28. During its evolution, three cyclones were found to be rooted in this structure. Each cyclone event lasted for about 8 to 10 hr. While near the polar region, another RNF was investigated. It lasted for a shorter time (∼70 hr), from 2013 July 7 to 9. There were two cyclones rooted in the RNF and each lasted for 8 and 11 hr, respectively. For the two given examples, the cyclones have a similar dynamic evolution, and thus we put forward a new term: homologous cyclones. The detected brightening in AIA 171 Å maps indicates the release of energy, which is potentially available to heat the corona.

  5. How homologous recombination maintains telomere integrity.

    PubMed

    Tacconi, Eliana M C; Tarsounas, Madalena

    2015-06-01

    Telomeres protect the ends of linear chromosomes against loss of genetic information and inappropriate processing as damaged DNA and are therefore crucial to the maintenance of chromosome integrity. In addition to providing a pathway for genome-wide DNA repair, homologous recombination (HR) plays a key role in telomere replication and capping. Consistent with this, the genomic instability characteristic of HR-deficient cells and tumours is driven in part by telomere dysfunction. Here, we discuss the mechanisms by which HR modulates the response to intrinsic cellular challenges that arise during telomere replication, as well as its impact on the assembly of telomere protective structures. How normal and tumour cells differ in their ability to maintain telomeres is deeply relevant to the search for treatments that would selectively eliminate cells whose capacity for HR-mediated repair has been compromised.

  6. Homologies among Coniferophyte cones: further observations

    NASA Astrophysics Data System (ADS)

    Grauvogel-Stamm, Léa; Galtier, Jean

    1998-04-01

    A reinvestigation of the Triassic conifer pollen cone of Darneya shows evidence that clusters of pollen sacs are attached (adnate), at regular intervals, to the upper side of the stalk and that the distribution of stomata is restricted to the apical part of the abaxial side of the peltate scale. These features and others, such as the commissure visible on the stalk and the scale, suggest a dual nature of the male scale complex of Darneya which therefore is interpreted as an abaxial bract fused with an adaxial fertile shoot bearing several clusters of pollen sacs. This conifer pollen cone is thus considered as a compound strobilus (inflorescence) homologous with the female cone of the conifers and therefore with the cones, both male and female, of the cordaites.

  7. Chatter detection in turning using persistent homology

    NASA Astrophysics Data System (ADS)

    Khasawneh, Firas A.; Munch, Elizabeth

    2016-03-01

    This paper describes a new approach for ascertaining the stability of stochastic dynamical systems in their parameter space by examining their time series using topological data analysis (TDA). We illustrate the approach using a nonlinear delayed model that describes the tool oscillations due to self-excited vibrations in turning. Each time series is generated using the Euler-Maruyama method and a corresponding point cloud is obtained using the Takens embedding. The point cloud can then be analyzed using a tool from TDA known as persistent homology. The results of this study show that the described approach can be used for analyzing datasets of delay dynamical systems generated both from numerical simulation and experimental data. The contributions of this paper include presenting for the first time a topological approach for investigating the stability of a class of nonlinear stochastic delay equations, and introducing a new application of TDA to machining processes.

  8. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

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

    1993-01-01

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

  9. Functional profiling of the Saccharomyces cerevisiae genome.

    PubMed

    Giaever, Guri; Chu, Angela M; Ni, Li; Connelly, Carla; Riles, Linda; Véronneau, Steeve; Dow, Sally; Lucau-Danila, Ankuta; Anderson, Keith; André, Bruno; Arkin, Adam P; Astromoff, Anna; El-Bakkoury, Mohamed; Bangham, Rhonda; Benito, Rocio; Brachat, Sophie; Campanaro, Stefano; Curtiss, Matt; Davis, Karen; Deutschbauer, Adam; Entian, Karl-Dieter; Flaherty, Patrick; Foury, Francoise; Garfinkel, David J; Gerstein, Mark; Gotte, Deanna; Güldener, Ulrich; Hegemann, Johannes H; Hempel, Svenja; Herman, Zelek; Jaramillo, Daniel F; Kelly, Diane E; Kelly, Steven L; Kötter, Peter; LaBonte, Darlene; Lamb, David C; Lan, Ning; Liang, Hong; Liao, Hong; Liu, Lucy; Luo, Chuanyun; Lussier, Marc; Mao, Rong; Menard, Patrice; Ooi, Siew Loon; Revuelta, Jose L; Roberts, Christopher J; Rose, Matthias; Ross-Macdonald, Petra; Scherens, Bart; Schimmack, Greg; Shafer, Brenda; Shoemaker, Daniel D; Sookhai-Mahadeo, Sharon; Storms, Reginald K; Strathern, Jeffrey N; Valle, Giorgio; Voet, Marleen; Volckaert, Guido; Wang, Ching-yun; Ward, Teresa R; Wilhelmy, Julie; Winzeler, Elizabeth A; Yang, Yonghong; Yen, Grace; Youngman, Elaine; Yu, Kexin; Bussey, Howard; Boeke, Jef D; Snyder, Michael; Philippsen, Peter; Davis, Ronald W; Johnston, Mark

    2002-07-25

    Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

  10. Sugar and Glycerol Transport in Saccharomyces cerevisiae.

    PubMed

    Bisson, Linda F; Fan, Qingwen; Walker, Gordon A

    2016-01-01

    In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition. PMID:26721273

  11. Sugar and Glycerol Transport in Saccharomyces cerevisiae.

    PubMed

    Bisson, Linda F; Fan, Qingwen; Walker, Gordon A

    2016-01-01

    In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition.

  12. Saccharomyces cerevisiae structural cell wall mannoprotein.

    PubMed

    Frevert, J; Ballou, C E

    1985-01-29

    A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material. PMID:3888262

  13. Copper transport in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Martinez, L.D.; Connelly, J.L.

    1987-05-01

    Biochemical processes involved in the movement of copper (Cu) into and out of the yeast Saccharomyces Cerevisiae have been investigated. Overall uptake of Cu was measured by disappearance of Cu from the reaction mixture by atomic absorption sensitive to 10/sup -10/M. The process of Cu influx is composed of a prerequisite binding and subsequent transport. The binding is non-energetic but is competitively inhibited by zinc(Zn). Transport is energetic as shown by an increased influx in the presence of added glucose. This process is prevented by 2,4-dinitrophenol(DNP). Cu influx is accompanied by an exchange for potassium(K) in a ratio of K:Cu=2:1. The process of Cu efflux involves a second type of binding site, probably of low affinity but large capacity. The presence of glucose causes the binding of extracellular Cu to these sites in a non-energy-dependent mechanism which prevents Cu efflux. Zn does not compete. DNP has no effect. The K:Cu ratio of 4:1 observed in the absence of glucose suggests a lowered net Cu uptake as a result of concomitant efflux activity. Finally, in the absence but not the presence of glucose, the pH of the extracellular solution increases. These observations are consistent with the idea that (a) yeast membrane has two Cu-binding sites, one of which participates in influx and one in efflux; (b) Cu exchanges with K during influx and with protons during efflux.

  14. Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

    PubMed Central

    Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

    1995-01-01

    Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

  15. Modeling Non-homologous End Joining

    NASA Technical Reports Server (NTRS)

    Li, Yongfeng

    2013-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed

  16. CIRCULAR RIBBON FLARES AND HOMOLOGOUS JETS

    SciTech Connect

    Wang Haimin; Liu Chang

    2012-12-01

    Solar flare emissions in the chromosphere often appear as elongated ribbons on both sides of the magnetic polarity inversion line (PIL), which has been regarded as evidence of a typical configuration of magnetic reconnection. However, flares having a circular ribbon have rarely been reported, although it is expected in the fan-spine magnetic topology involving reconnection at a three-dimensional (3D) coronal null point. We present five circular ribbon flares with associated surges, using high-resolution and high-cadence H{alpha} blue wing observations obtained from the recently digitized films of Big Bear Solar Observatory. In all the events, a central parasitic magnetic field is encompassed by the opposite polarity, forming a circular PIL traced by filament material. Consequently, a flare kernel at the center is surrounded by a circular flare ribbon. The four homologous jet-related flares on 1991 March 17 and 18 are of particular interest, as (1) the circular ribbons brighten sequentially, with cospatial surges, rather than simultaneously, (2) the central flare kernels show an intriguing 'round-trip' motion and become elongated, and (3) remote brightenings occur at a region with the same magnetic polarity as the central parasitic field and are co-temporal with a separate phase of flare emissions. In another flare on 1991 February 25, the circular flare emission and surge activity occur successively, and the event could be associated with magnetic flux cancellation across the circular PIL. We discuss the implications of these observations combining circular flare ribbons, homologous jets, and remote brightenings for understanding the dynamics of 3D magnetic restructuring.

  17. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: A conserved structure with an essential function

    SciTech Connect

    Allison, L.A.; Wong, J.K.C.; Fitzpatrick, V.D.; Moyle, M.; Ingles, C.J.

    1988-01-01

    Using DNA encoding the largest subunit of Drosophila melanogaster RNA polymerase II, the authors isolated the homologous hamster RPO21 gene. Nucleotide sequencing of both the hamster and D. melanogaster RPO21 DNAs confirmed that the RPO21 polypeptides of these two species, like the Saccharomyces cerevisiae RPO21 polypeptide, contain both an N-terminal region homologous to the Escherichia coli RNA polymerase subunit ..beta..' and a unique polymerase II-specific C-terminal domain. This C-terminal domain, encoded by separate exons in the D. melanogaster and hamster genes, consists of a tandemly repeated heptapeptide sequence. By constructing a series of deletions in DNA encoding the 26 heptapeptide repeats normally present in the S. cerevisiae RPO21 polypeptide, the authors have established that a minimum of between 9 and 11 repeats is necessary for RPO21 function in yeast cells. Replacement of the yeast RPO21 heptapeptide repeats by the longer hamster repetitive domain resulted in viable yeast cells with no detectable mutant phenotype, while a similar replacement of the yeast repeats by the more divergent D. melanogaster repeats was a recessive lethal mutation. The authors suggest that this novel repetitive domain is essential for proper initiation of transcription by RNA polymerase II and that it may mediate the functions of TATA boxes, upstream activating sequences, and enhancers.

  18. Sgs1, a Homologue of the Bloom's and Werner's Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

    PubMed Central

    Watt, P. M.; Hickson, I. D.; Borts, R. H.; Louis, E. J.

    1996-01-01

    The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae. PMID:8913739

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

    PubMed

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

    2012-08-01

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

  20. Ultrastructural changes of Saccharomyces cerevisiae in response to ethanol stress.

    PubMed

    Ma, Manli; Han, Pei; Zhang, Ruimin; Li, Hao

    2013-09-01

    In the fermentative process using Saccharomyces cerevisiae to produce bioethanol, the performance of cells is often compromised by the accumulation of ethanol. However, the mechanism of how S. cerevisiae responds against ethanol stress remains elusive. In the current study, S. cerevisiae cells were cultured in YPD (yeast extract - peptone - dextrose) medium containing various concentrations of ethanol (0%, 2.5%, 5%, 7.5%, 10%, and 15% (v/v)). Compared with the control group without ethanol, the mean cell volume of S. cerevisiae decreased significantly in the presence of 7.5% and 10% ethanol after incubation for 16 h (P < 0.05), and in the presence of 15% ethanol at all 3 sampling time points (1, 8, and 16 h) (P < 0.05). The exposure of S. cerevisiae cells to ethanol also led to an increase in malonyldialdehyde content (P < 0.05) and a decrease in sulfhydryl group content (P < 0.05). Moreover, the observations through transmission electron microscopy enabled us to relate ultrastructural changes elicited by ethanol with the cellular stress physiology. Under ethanol stress, the integrity of the cell membrane was compromised. The swelling or distortion of mitochondria together with the occurrence of a single and large vacuole was correlated with the addition of ethanol. These results suggested that the cell membrane is one of the targets of ethanol, and the degeneration of mitochondria promoted the accumulation of intracellular reactive oxygen species.

  1. Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae.

    PubMed

    Villas-Bôas, Silas Granato; Kesson, Mats; Nielsen, Jens

    2005-05-01

    Glyoxylate biosynthesis in Saccharomyces cerevisiae is traditionally mainly ascribed to the reaction catalyzed by isocitrate lyase (Icl), which converts isocitrate to glyoxylate and succinate. However, Icl is generally reported to be repressed by glucose and yet glyoxylate is detected at high levels in S. cerevisiae extracts during cultivation on glucose. In bacteria there is an alternative pathway for glyoxylate biosynthesis that involves a direct oxidation of glycine. Therefore, we investigated the glycine metabolism in S. cerevisiae coupling metabolomics data and (13)C-isotope-labeling analysis of two reference strains and a mutant with a deletion in a gene encoding an alanine:glyoxylate aminotransferase. The strains were cultivated on minimal medium containing glucose or galactose, and (13)C-glycine as sole nitrogen source. Glyoxylate presented (13)C-labeling in all cultivation conditions. Furthermore, glyoxylate seemed to be converted to 2-oxovalerate, an unusual metabolite in S. cerevisiae. 2-Oxovalerate can possibly be converted to 2-oxoisovalerate, a key precursor in the biosynthesis of branched-chain amino acids. Hence, we propose a new pathway for glycine catabolism and glyoxylate biosynthesis in S. cerevisiae that seems not to be repressed by glucose and is active under both aerobic and anaerobic conditions. This work demonstrates the great potential of coupling metabolomics data and isotope-labeling analysis for pathway reconstructions.

  2. SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family

    SciTech Connect

    Lapenta, V.; Chiurazzi, P.; Van Der Spek, P.; Hanaoka, Fumio

    1997-03-01

    cDNA selection was used to isolate coding sequences from cosmids mapping to the gene-rich telomeric region of human chromosome 21q. A novel cDNA, termed SMT3A, was isolated and mapped between the loci PFKL and D21S171, about 2.2 Mb proximal to the telomere. The predicted protein of 103 amino acids appears to be a homologue of the Saccharomyces cerevisiae SMT3 protein, whose gene was previously isolated as a suppressor of mutations in the MIF2 gene. The yeast MIF2 gene encodes an essential centromeric protein and shows homology to mammalian CENP-C, an integral component of active kinetochores. SMT3A was found to be highly homologous to two other recently isolated human genes, suggesting the presence of a new gene family. Homologous sequences were also found in protozoa, metazoa, and plants. Moreover, all predicted proteins show significant homology to ubiquitin. The proposed role of yeast SMT3 as centromeric protein and the strong evolutionary conservation of the SMT3A gene suggest an involvement of the encoded protein in the function and/or structure of the eukaryotic kinetochore. 30 refs., 5 figs.

  3. Saccharomyces cerevisiae Dmc1 and Rad51 proteins preferentially function with Tid1 and Rad54 proteins, respectively, to promote DNA strand invasion during genetic recombination.

    PubMed

    Nimonkar, Amitabh V; Dombrowski, Christopher C; Siino, Joseph S; Stasiak, Alicja Z; Stasiak, Andrzej; Kowalczykowski, Stephen C

    2012-08-17

    The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

  4. Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.

    PubMed

    Kaps, Sonja; Kettner, Karina; Migotti, Rebekka; Kanashova, Tamara; Krause, Udo; Rödel, Gerhard; Dittmar, Gunnar; Kriegel, Thomas M

    2015-03-01

    The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.

  5. Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation*

    PubMed Central

    Kaps, Sonja; Kettner, Karina; Migotti, Rebekka; Kanashova, Tamara; Krause, Udo; Rödel, Gerhard; Dittmar, Gunnar; Kriegel, Thomas M.

    2015-01-01

    The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains. PMID:25593311

  6. Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration.

    PubMed

    van Attikum, H; Bundock, P; Hooykaas, P J

    2001-11-15

    Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements. PMID:11707425

  7. Real-time analysis of double-strand DNA break repair by homologous recombination

    PubMed Central

    Hicks, Wade M.; Yamaguchi, Miyuki; Haber, James E.

    2011-01-01

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion. PMID:21292986

  8. Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

    PubMed Central

    Clement, Sarah T.; Dixit, Gauri; Dohlman, Henrik G.

    2014-01-01

    Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior. PMID:24003255

  9. Real-time analysis of double-strand DNA break repair by homologous recombination.

    PubMed

    Hicks, Wade M; Yamaguchi, Miyuki; Haber, James E

    2011-02-22

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.

  10. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

    PubMed

    Loraine, A E; Yalovsky, S; Fabry, S; Gruissem, W

    1996-04-01

    Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.

  11. Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Clancy, M J; Buten-Magee, B; Straight, D J; Kennedy, A L; Partridge, R M; Magee, P T

    1983-01-01

    A library of Saccharomyces cerevisiae DNA in the vector lambda Charon 28 was probed for sequences complementary to cDNA made from poly(A)+ RNA isolated from the well-sporulating yeast strain AP1 a/alpha. The RNA was isolated from cells that had been incubated 7, 9, 11, and 13 hr in sporulation medium. DNA complementary to poly(A)+ RNA from alpha/alpha(nonsporulating) AP1 was used as a control, and 46 bacteriophage that gave a stronger response with a/alpha cDNA than with alpha/alpha cDNA were obtained in a screening of three yeast genomes worth of DNA. Two of the bacteriophage appeared to contain a/alpha-specific genes, in that they hybridized to cDNA from vegetative a/alpha RNA. The rest appeared to correspond to a/alpha genes expressed preferentially during sporulation. Restriction endonuclease analysis of four of the cloned sequences revealed a single major region of transcription in each; these regions ranged in size from 2.5 to 4.0 kilobases. RNA blot analysis showed that, in three of the four cases, transcripts of two different sizes were homologous to the cloned sequence. In all four cases, the homologous transcripts appeared at about 7 hr and were decreasing in amount by 13 hr. These results provide evidence for transcriptional control of genes expressed during sporulation and for at least one group of genes that is turned on at about the time of meiosis I in sporulation. Images PMID:6304689

  12. [Homologous recombination among bacterial genomes: the measurement and identification].

    PubMed

    Xianwei, Yang; Ruifu, Yang; Yujun, Cui

    2016-02-01

    Homologous recombination is one of important sources in shaping the bacterial population diversity, which disrupts the clonal relationship among different lineages through horizontal transferring of DNA-segments. As consequence of blurring the vertical inheritance signals, the homologous recombination raises difficulties in phylogenetic analysis and reconstruction of population structure. Here we discuss the impacts of homologous recombination in inferring phylogenetic relationship among bacterial isolates, and summarize the tools and models separately used in recombination measurement and identification. We also highlight the merits and drawbacks of various approaches, aiming to assist in the practical application for the analysis of homologous recombination in bacterial evolution research. PMID:26907777

  13. Cadmium-induced oxidative stress in Saccharomyces cerevisiae.

    PubMed

    Muthukumar, Kannan; Nachiappan, Vasanthi

    2010-12-01

    The present study was undertaken to determine the effect of cadmium (Cd) on the antioxidant status of the yeast Saccharomyces cerevisiae. S. cerevisiae serves as a good eukaryotic model system for the study of the molecular mechanisms of oxidative stress. We investigated the adaptative response of S. cerevisiae exposed to Cd. Yeast cells could tolerate up to 100 microM Cd and an inhibition in the growth and viability was observed. Exposure of yeast cells to Cd showed an increase in malondialdehyde and glutathione. The activities of catalase, superoxide dismutase and glutathione peroxidase were also high in Cd-exposed cells. The incorporation of Cd led to significant increase in iron, zinc and inversely the calcium, copper levels were reduced. The results suggest that antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumably, these enzymes are essential for counteracting the pro-oxidant effects of Cd. PMID:21355423

  14. Phosphate transport and sensing in Saccharomyces cerevisiae.

    PubMed Central

    Wykoff, D D; O'Shea, E K

    2001-01-01

    Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters. PMID:11779791

  15. Regulation of Cation Balance in Saccharomyces cerevisiae

    PubMed Central

    Cyert, Martha S.; Philpott, Caroline C.

    2013-01-01

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

  16. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.

    PubMed Central

    Gangloff, S; McDonald, J P; Bendixen, C; Arthur, L; Rothstein, R

    1994-01-01

    We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgs1 (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgs1 protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgs1 creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgs1 helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide. Images PMID:7969174

  17. Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae.

    PubMed

    Fišerová, Jindřiška; Richardson, Christine; Goldberg, Martin W

    2016-01-01

    Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events. PMID:27515085

  18. A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae.

    PubMed Central

    de los Santos, T; Loidl, J; Larkin, B; Hollingsworth, N M

    2001-01-01

    The MMS4 gene of Saccharomyces cerevisiae was originally identified due to its sensitivity to MMS in vegetative cells. Subsequent studies have confirmed a role for MMS4 in DNA metabolism of vegetative cells. In addition, mms4 diploids were observed to sporulate poorly. This work demonstrates that the mms4 sporulation defect is due to triggering of the meiotic recombination checkpoint. Genetic, physical, and cytological analyses suggest that MMS4 functions after the single end invasion step of meiotic recombination. In spo13 diploids, red1, but not mek1, is epistatic to mms4 for sporulation and spore viability, suggesting that MMS4 may be required only when homologs are capable of undergoing synapsis. MMS4 and MUS81 are in the same epistasis group for spore viability, consistent with biochemical data that show that the two proteins function in a complex. In contrast, MMS4 functions independently of MSH5 in the production of viable spores. We propose that MMS4 is required for the processing of specific recombination intermediates during meiosis. PMID:11779793

  19. Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway.

    PubMed Central

    Shinohara, Miki; Sakai, Kazuko; Shinohara, Akira; Bishop, Douglas K

    2003-01-01

    Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination. PMID:12702674

  20. Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Soustelle, Christine; Vedel, Michèle; Kolodner, Richard; Nicolas, Alain

    2002-01-01

    In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis. PMID:12072452

  1. Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion

    PubMed Central

    Kiechle, Markus; Manivasakam, Palaniyandi; Eckardt-Schupp, Friederike; Schiestl, Robert H.; Friedl, Anna A.

    2002-01-01

    Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by γ-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30°C, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements. PMID:12490727

  2. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.

    PubMed

    Athenstaedt, K; Zweytick, D; Jandrositz, A; Kohlwein, S D; Daum, G

    1999-10-01

    Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.

  3. Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Athenstaedt, Karin; Zweytick, Dagmar; Jandrositz, Anita; Kohlwein, Sepp Dieter; Daum, Günther

    1999-01-01

    Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism. PMID:10515935

  4. Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

    PubMed Central

    Velours, J; Esparza, M; Hoppe, J; Sebald, W; Guerin, B

    1984-01-01

    The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans. Images Fig. 2. PMID:6323165

  5. Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

    PubMed Central

    Corey, E J; Matsuda, S P; Bartel, B

    1994-01-01

    We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter. Images PMID:8134375

  6. Non-repair pathways for minimizing protein isoaspartyl damage in the yeast Saccharomyces cerevisiae.

    PubMed

    Patananan, Alexander N; Capri, Joseph; Whitelegge, Julian P; Clarke, Steven G

    2014-06-13

    The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.

  7. NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

    PubMed

    Higuchi, Naoki; Ito, Yasuhiro; Kato, Jun; Ogihara, Jun; Kasumi, Takafumi

    2016-06-01

    Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent.

  8. Isolation and characterization of a thiamin transport gene, THI10, from Saccharomyces cerevisiae.

    PubMed

    Enjo, F; Nosaka, K; Ogata, M; Iwashima, A; Nishimura, H

    1997-08-01

    We isolated a thiamin transporter gene, THI10, from a genomic library of Saccharomyces cerevisiae by the complementation of a yeast mutant defective in thiamin transport activity. The THI10 gene contained an open reading frame of 1,794 base pairs encoding a 598-amino acid polypeptide with a calculated molecular weight of 66, 903. The nucleotide sequence of THI10 is completely identical to that of an anonymous DNA (open reading frame L8083.2) mapped to chromosome XII; two other genes (open reading frames YOR071c and YOR192c) in chromosome XV are extremely similar to THI10. Moreover, the THI10 gene product showed significant sequence homology with yeast allantoin and uracil transporters. Hydropathy profile suggested that THI10 product is highly hydrophobic and contains many transmembrane regions. Gene disruption of the THI10 locus completely abolished the thiamin transport activity and thiamin binding activity in yeast plasma membrane fraction. Both the transport and thiamin binding activities were restored in the disrupted cells when the THI10 open reading frame was expressed by yeast GAL1 promoter, suggesting that the THI10 gene encodes for the thiamin transport carrier protein. Northern blot analysis demonstrated that THI10 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and that it requires a positive regulatory factor encoded by THI3 gene.

  9. Ssh4, Rcr2 and Rcr1 affect plasma membrane transporter activity in Saccharomyces cerevisiae.

    PubMed

    Kota, Jhansi; Melin-Larsson, Monika; Ljungdahl, Per O; Forsberg, Hanna

    2007-04-01

    Nutrient uptake in the yeast Saccharomyces cerevisiae is a highly regulated process. Cells adjust levels of nutrient transporters within the plasma membrane at multiple stages of the secretory and endosomal pathways. In the absence of the ER-membrane-localized chaperone Shr3, amino acid permeases (AAP) inefficiently fold and are largely retained in the ER. Consequently, shr3 null mutants exhibit greatly reduced rates of amino acid uptake due to lower levels of AAPs in their plasma membranes. To further our understanding of mechanisms affecting AAP localization, we identified SSH4 and RCR2 as high-copy suppressors of shr3 null mutations. The overexpression of SSH4, RCR2, or the RCR2 homolog RCR1 increases steady-state AAP levels, whereas the genetic inactivation of these genes reduces steady-state AAP levels. Additionally, the overexpression of any of these suppressor genes exerts a positive effect on phosphate and uracil uptake systems. Ssh4 and Rcr2 primarily localize to structures associated with the vacuole; however, Rcr2 also localizes to endosome-like vesicles. Our findings are consistent with a model in which Ssh4, Rcr2, and presumably Rcr1, function within the endosome-vacuole trafficking pathway, where they affect events that determine whether plasma membrane proteins are degraded or routed to the plasma membrane.

  10. Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis.

    PubMed Central

    Ronne, H; Carlberg, M; Hu, G Z; Nehlin, J O

    1991-01-01

    We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles. Images PMID:1656215

  11. Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

    PubMed Central

    Thill, G P; Kramer, R A; Turner, K J; Bostian, K A

    1983-01-01

    The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion. Images PMID:6343840

  12. The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

    PubMed Central

    Weber, V; Harata, M; Hauser, H; Wintersberger, U

    1995-01-01

    Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin. Images PMID:8573785

  13. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  14. Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

    PubMed Central

    Neiman, A. M.; Mhaiskar, V.; Manus, V.; Galibert, F.; Dean, N.

    1997-01-01

    The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. PMID:9055074

  15. Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae.

    PubMed

    Schirmaier, F; Philippsen, P

    1984-12-20

    The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2. Both genes were cloned. TEF1 maps on chromosome II close to LYS2. The location of TEF2 is unknown. TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1. TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids. The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence. This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia. A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes. PMID:6396088

  16. NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

    PubMed

    Higuchi, Naoki; Ito, Yasuhiro; Kato, Jun; Ogihara, Jun; Kasumi, Takafumi

    2016-06-01

    Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent. PMID:26589784

  17. High speed homology search with FPGAs.

    PubMed

    Yamaguchi, Yoshiki; Maruyama, Tsutomu; Konagaya, Akihiko

    2002-01-01

    We will introduce a way how we can achieve high speed homology search by only adding one off-the-shelf PCI board with one Field Programmable Gate Array (FPGA) to a Pentium based computer system in use. FPGA is a reconfigurable device, and any kind of circuits, such as pattern matching program, can be realized in a moment. The performance is almost proportional to the size of FPGA which is used in the system, and FPGAs are becoming larger and larger following Moore's law. We can easily obtain latest/larger FPGAs in the form off-the-shelf PCI boards with FPGAs, at low costs. The result which we obtained is as follows. The performance is most comparable with small to middle class dedicated hardware systems when we use a board with one of the latest FPGAs and the performance can be furthermore accelerated by using more number of FPGA boards. The time for comparing a query sequence of 2,048 elements with a database sequence of 64 million elements by the Smith-Waterman algorithm is about 34 sec, which is about 330 times faster than a desktop computer with a 1 GHz Pentium III. We can also accelerate the performance of a laptop computer using a PC card with one smaller FPGA. The time for comparing a query sequence (1,024) with the database sequence (64 million) is about 185 sec, which is about 30 times faster than the desktop computer.

  18. CBH1 homologs and varian CBH1 cellulase

    SciTech Connect

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2014-07-01

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  19. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2008-11-18

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  20. CBH1 homologs and variant CBH1 cellulases

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2011-05-31

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  1. Uncoupling of 3'-phosphatase and 5'-kinase functions in budding yeast. Characterization of Saccharomyces cerevisiae DNA 3'-phosphatase (TPP1).

    PubMed

    Vance, J R; Wilson, T E

    2001-05-01

    Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

  2. Multiple overlapping homologies between two rheumatoid antigens and immunosuppressive viruses.

    PubMed Central

    Douvas, A; Sobelman, S

    1991-01-01

    Amino acid (aa) sequence homologies between viruses and autoimmune nuclear antigens are suggestive of viral involvement in disorders such as systemic lupus erythematosus (SLE) and scleroderma. We analyzed the frequency of exact homologies of greater than or equal to 5 aa between 61 viral proteins (19,827 aa), 8 nuclear antigens (3813 aa), and 41 control proteins (11,743 aa). Both pentamer and hexamer homologies between control proteins and viruses are unexpectedly abundant, with hexamer matches occurring in 1 of 3 control proteins (or once every 769 aa). However, 2 nuclear antigens, the SLE-associated 70-kDa antigen and the scleroderma-associated CENP-B protein, are highly unusual in containing multiple homologies to a group of synergizing immunosuppressive viruses. Two viruses, herpes simplex virus 1 (HSV-1) and human immunodeficiency virus 1 (HIV-1), contain sequences exactly duplicated at 15 sites in the 70-kDa antigen and at 10 sites in CENP-B protein. The immediate-early (IE) protein of HSV-1, which activates HIV-1 regulatory functions, contains three homologies to the 70-kDa antigen (two hexamers and a pentamer) and two to CENP-B (a hexamer and pentamer). There are four homologies (including a hexamer) common to the 70-kDa antigen and Epstein-Barr virus, and three homologies (including two hexamers) common to CENP-B and cytomegalovirus. The majority of homologies in both nuclear antigens are clustered in highly charged C-terminal domains containing epitopes for human autoantibodies. Furthermore, most homologies have a contiguous or overlapping distribution, thereby creating a high density of potential epitopes. In addition to the exact homologies tabulated, motifs of matching sequences are repeated frequently in these domains. Our analysis suggests that coexpression of heterologous viruses having common immunosuppressive functions may generate autoantibodies cross-reacting with certain nuclear proteins. PMID:1712488

  3. Peridinialean dinoflagellate plate patterns, labels and homologies

    USGS Publications Warehouse

    Edwards, L.E.

    1990-01-01

    Tabulation patterns for peridinialean dinoflagellate thecae and cysts have been traditionally expressed using a plate labelling system described by C.A. Kofoid in the early 1900's. This system can obscure dinoflagellate plate homologies and has not always been strictly applied. The plate-labelling system presented here introduces new series labels but incorporates key features and ideas from the more recently proposed systems of G.L. Eaton and F.J.R. Taylor, as modified by W.R. Evitt. Plate-series recognition begins with the cingulum (C-series) and proceeds from the cingulum toward the apex for the three series of the epitheca/epicyst and proceeds from the cingulum toward the antapex for the two series of the hypotheca/hypocyst. The epithecal/epicystal model consists of eight plates that touch the anterior margin of the cingulum (E-series: plates E1-E7, ES), seven plates toward the apex that touch the E-series plates (M-series: R, M1-M6), and up to seven plates near the apex that do not touch E-series plates (D-series: Dp-Dv). The hypothecal/hypocystal model consists of eight plates that touch the posterior margin of the cingulum (H-series: H1-H6,HR,HS) and three plates toward the antapex (T1-T3). Epithecal/epicystal tabulation patterns come in both 8- and 7- models, corresponding to eight and seven plates, respectively, in the E-series. Hypothecal/hypocystal tabulation patterns also come in both 8- and 7-models, corresponding to eight and seven plates, respectively, in the H-series. By convention, the 7-model epitheca/epicyst has no plates E1 and M1; the 7-model hypotheca/hypocyst has no plate H6. Within an 8-model or 7-model, the system emphasizes plates that are presumed to be homologous by giving them identical labels. I introduce the adjectives "monothigmate", "dithigmate," and "trithigmate" to designate plates touching one, two, and three plates, respectively, of the adjacent series. The term "thigmation" applies to the analysis of plate contacts between

  4. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism.

    PubMed

    Kim, Soo Rin; Park, Yong-Cheol; Jin, Yong-Su; Seo, Jin-Ho

    2013-11-01

    Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism. PMID:23524005

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

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

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

  6. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

  8. The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

    PubMed

    Johnston, M; Hillier, L; Riles, L; Albermann, K; André, B; Ansorge, W; Benes, V; Brückner, M; Delius, H; Dubois, E; Düsterhöft, A; Entian, K D; Floeth, M; Goffeau, A; Hebling, U; Heumann, K; Heuss-Neitzel, D; Hilbert, H; Hilger, F; Kleine, K; Kötter, P; Louis, E J; Messenguy, F; Mewes, H W; Hoheisel, J D

    1997-05-29

    The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.

  9. No current evidence for widespread dosage compensation in S. cerevisiae

    PubMed Central

    Torres, Eduardo M; Springer, Michael; Amon, Angelika

    2016-01-01

    Previous studies of laboratory strains of budding yeast had shown that when gene copy number is altered experimentally, RNA levels generally scale accordingly. This is true when the copy number of individual genes or entire chromosomes is altered. In a recent study, Hose et al. (2015) reported that this tight correlation between gene copy number and RNA levels is not observed in recently isolated wild Saccharomyces cerevisiae variants. To understand the origins of this proposed difference in gene expression regulation between natural variants and laboratory strains of S. cerevisiae, we evaluated the karyotype and gene expression studies performed by Hose et al. on wild S. cerevisiae strains. In contrast to the results of Hose et al., our reexamination of their data revealed a tight correlation between gene copy number and gene expression. We conclude that widespread dosage compensation occurs neither in laboratory strains nor in natural variants of S. cerevisiae. DOI: http://dx.doi.org/10.7554/eLife.10996.001 PMID:26949255

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

    PubMed

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

    2016-02-01

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

  11. 2μ plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

    PubMed

    Strope, Pooja K; Kozmin, Stanislav G; Skelly, Daniel A; Magwene, Paul M; Dietrich, Fred S; McCusker, John H

    2015-12-01

    We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer.

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

    PubMed

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

    2014-04-14

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

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

    PubMed

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

    2001-10-01

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

  14. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Homologous recombination in bovine pestiviruses. Phylogenetic and statistic evidence.

    PubMed

    Jones, Leandro Roberto; Weber, E Laura

    2004-12-01

    Bovine pestiviruses (Bovine Viral Diarrea Virus 1 (BVDV 1) and Bovine Viral Diarrea Virus 2 (BVDV 2)) belong to the genus Pestivirus (Flaviviridae), which is composed of positive stranded RNA viruses causing significant economic losses world-wide. We used phylogenetic and bootstrap analyses to systematically scan alignments of previously sequenced genomes in order to explore further the evolutionary mechanisms responsible for variation in the virus. Previously published data suggested that homologous crossover might be one of the mechanisms responsible for the genomic rearrangements observed in cytopathic (cp) strains of bovine pestiviruses. Nevertheless, homologous recombination involves not just homologous crossovers, but also replacement of a homologous region of the acceptor RNA. Furthermore, cytopathic strains represent dead paths in evolution, since they are isolated exclusively from the fatal cases of mucosal disease. Herein, we report evidence of homologous inter-genotype recombination in the genome of a non-cytopathic (ncp) strain of Bovine Viral Diarrea Virus 1, the type species of the genus Pestivirus. We also show that intra-genotype homologous recombination might be a common phenomenon in both species of Pestivirus. This evidence demonstrates that homologous recombination contribute to the diversification of bovine pestiviruses in nature. Implications for virus evolution, taxonomy and phylogenetics are discussed.

  16. Linking yeast genetics to mammalian genomes: identification and mapping of the human homolog of CDC27 via the expressed sequence tag (EST) data base.

    PubMed Central

    Tugendreich, S; Boguski, M S; Seldin, M S; Hieter, P

    1993-01-01

    We describe a strategy for quickly identifying and positionally mapping human homologs of yeast genes to cross-reference the biological and genetic information known about yeast genes to mammalian chromosomal maps. Optimized computer search methods have been developed to scan the rapidly expanding expressed sequence tag (EST) data base to find human open reading frames related to yeast protein sequence queries. These methods take advantage of the newly developed BLOSUM scoring matrices and the query masking function SEG. The corresponding human cDNA is then used to obtain a high-resolution map position on human and mouse chromosomes, providing the links between yeast genetic analysis and mapped mammalian loci. By using these methods, a human homolog of Saccharomyces cerevisiae CDC27 has been identified and mapped to human chromosome 17 and mouse chromosome 11 between the Pkca and Erbb-2 genes. Human CDC27 encodes an 823-aa protein with global similarity to its fungal homologs CDC27, nuc2+, and BimA. Comprehensive cross-referencing of genes and mutant phenotypes described in humans, mice, and yeast should accelerate the study of normal eukaryotic biology and human disease states. Images Fig. 2 PMID:8234252

  17. The history of the homology concept and the "Phylogenetisches Symposium".

    PubMed

    Hossfeld, Uwe; Olsson, Lennart

    2005-11-01

    The homology concept has had a long and varied history, starting out as a geometrical term in ancient Greece. Here we describe briefly how a typological use of homology to designate organs and body parts in the same position anatomically in different organisms was changed by Darwin's theory of evolution into a phylogenetic concept. We try to indicate the diversity of opinions on how to define and test for homology that has prevailed historically, before the important books by Hennig (1950. Grundzüge einer Theorie der Phylogenetischen Systematik. Deutscher Zentralverlag, Berlin) and Remane (1952. Die Grundlagen des Natürlichen Systems, der Vergleichenden Anatomie und der Phylogenetik. Geest & Portig, Leipzig) brought more rigor into both the debate on homology and into the usage of the term homology among systematists. Homology as a theme has recurred repeatedly throughout the history of the "Phylogenetisches Symposium" and we give a very brief overview of the different aspects of homology that have been discussed at specific symposia over the last 48 years. We also honour the fact that the 2004 symposium was held in Jena by pointing to the roles played by biologists active in Jena, such as Ernst Haeckel and Carl Gegenbaur, in starting the development towards a homology concept concordant with an evolutionary world view. As historians of biology, we emphasize the importance of major treatises on homology and its history that may be little read by systematists active today, and have sometimes also received less attention by historians of biology than they deserve. Prominent among these are the works of Dietrich Starck, who also happened to be both a student, and later a benefactor, of systematics at Jena University. PMID:17046358

  18. The history of the homology concept and the "Phylogenetisches Symposium".

    PubMed

    Hossfeld, Uwe; Olsson, Lennart

    2005-11-01

    The homology concept has had a long and varied history, starting out as a geometrical term in ancient Greece. Here we describe briefly how a typological use of homology to designate organs and body parts in the same position anatomically in different organisms was changed by Darwin's theory of evolution into a phylogenetic concept. We try to indicate the diversity of opinions on how to define and test for homology that has prevailed historically, before the important books by Hennig (1950. Grundzüge einer Theorie der Phylogenetischen Systematik. Deutscher Zentralverlag, Berlin) and Remane (1952. Die Grundlagen des Natürlichen Systems, der Vergleichenden Anatomie und der Phylogenetik. Geest & Portig, Leipzig) brought more rigor into both the debate on homology and into the usage of the term homology among systematists. Homology as a theme has recurred repeatedly throughout the history of the "Phylogenetisches Symposium" and we give a very brief overview of the different aspects of homology that have been discussed at specific symposia over the last 48 years. We also honour the fact that the 2004 symposium was held in Jena by pointing to the roles played by biologists active in Jena, such as Ernst Haeckel and Carl Gegenbaur, in starting the development towards a homology concept concordant with an evolutionary world view. As historians of biology, we emphasize the importance of major treatises on homology and its history that may be little read by systematists active today, and have sometimes also received less attention by historians of biology than they deserve. Prominent among these are the works of Dietrich Starck, who also happened to be both a student, and later a benefactor, of systematics at Jena University.

  19. Benchmarking the next generation of homology inference tools

    PubMed Central

    Saripella, Ganapathi Varma; Sonnhammer, Erik L. L.; Forslund, Kristoffer

    2016-01-01

    Motivation: Over the last decades, vast numbers of sequences were deposited in public databases. Bioinformatics tools allow homology and consequently functional inference for these sequences. New profile-based homology search tools have been introduced, allowing reliable detection of remote homologs, but have not been systematically benchmarked. To provide such a comparison, which can guide bioinformatics workflows, we extend and apply our previously developed benchmark approach to evaluate the ‘next generation’ of profile-based approaches, including CS-BLAST, HHSEARCH and PHMMER, in comparison with the non-profile based search tools NCBI-BLAST, USEARCH, UBLAST and FASTA. Method: We generated challenging benchmark datasets based on protein domain architectures within either the PFAM + Clan, SCOP/Superfamily or CATH/Gene3D domain definition schemes. From each dataset, homologous and non-homologous protein pairs were aligned using each tool, and standard performance metrics calculated. We further measured congruence of domain architecture assignments in the three domain databases. Results: CSBLAST and PHMMER had overall highest accuracy. FASTA, UBLAST and USEARCH showed large trade-offs of accuracy for speed optimization. Conclusion: Profile methods are superior at inferring remote homologs but the difference in accuracy between methods is relatively small. PHMMER and CSBLAST stand out with the highest accuracy, yet still at a reasonable computational cost. Additionally, we show that less than 0.1% of Swiss-Prot protein pairs considered homologous by one database are considered non-homologous by another, implying that these classifications represent equivalent underlying biological phenomena, differing mostly in coverage and granularity. Availability and Implementation: Benchmark datasets and all scripts are placed at (http://sonnhammer.org/download/Homology_benchmark). Contact: forslund@embl.de Supplementary information: Supplementary data are available at

  20. Homology Groups of High-Resolution Temporal Rainfall

    NASA Astrophysics Data System (ADS)

    Vásquez Aguilar, R.; Carsteanu, A. A.

    2015-12-01

    Using high-resolution temporal rainfall intensities from Iowa City, IA (IIHR, U of Iowa), we perform an analysis of the homology groups generated by data connectivity in state space, and attempt a qualitative interpretation of the first and second homology groups. Let us note that homology groups are generated, in the context of topological data analysis (TDA), by representing the data in n-dimensional state space and building a connectivity diagram according to the respective distances between the data points. Subsequently, the topological invariants of the resulting connected structures are being analyzed.

  1. Importing the homology concept from biology into developmental psychology.

    PubMed

    Moore, David S

    2013-01-01

    To help introduce the idea of homology into developmental psychology, this article presents some of the concepts, distinctions, and guidelines biologists and philosophers of biology have devised to study homology. Some unresolved issues related to this idea are considered as well. Because homology reflects continuity across time, developmental scientists should find this concept to be useful in the study of psychological/behavioral development, just as biologists have found it essential in the study of the evolution and development of morphological and other characteristics.

  2. All Stable Characteristic Classes of Homological Vector Fields

    NASA Astrophysics Data System (ADS)

    Mosman, Elena; Sharapov, Alexey

    2010-12-01

    An odd vector field Q on a supermanifold M is called homological, if Q 2 = 0. The operator of Lie derivative L Q makes the algebra of smooth tensor fields on M into a differential tensor algebra. In this paper, we give a complete classification of certain invariants of homological vector fields called characteristic classes. These take values in the cohomology of the operator L Q and are represented by Q-invariant tensors made up of the homological vector field and a symmetric connection on M by means of the algebraic tensor operations and covariant differentiation.

  3. What is "homology thinking" and what is it for?

    PubMed

    Wagner, Günter P

    2016-01-01

    In this paper I examine the thesis by Marc Ereshefsky that, in evolutionary biology, there is a third style of thinking, besides the well-known "population thinking" and "tree thinking." Ereshefsky proposes "homology thinking" as a third approach, focused on the transformation of organismal phenotypes. In this short commentary, I aim at identifying the underlying biological assumptions for homology thinking and how they can be put to work in a research program within evolutionary biology. I propose that homology thinking is based on three insights: 1) multicellular organisms consist of developmentally individualized parts (sub-systems); 2) that developmental individuation entails evolutionary individuation, that is, variational quasi-independence; and 3) these individuated body parts are inherited, though indirectly, and form lineages that are recognized as homologies. These facts support a research program focused on the modification and origination of individuated body parts that supplements and puts into perspective the population genetic and phylogenetic approaches to the study of evolution. PMID:26486321

  4. Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

    PubMed Central

    2012-01-01

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

  5. MRFalign: protein homology detection through alignment of Markov random fields.

    PubMed

    Ma, Jianzhu; Wang, Sheng; Wang, Zhiyong; Xu, Jinbo

    2014-03-01

    Sequence-based protein homology detection has been extensively studied and so far the most sensitive method is based upon comparison of protein sequence profiles, which are derived from multiple sequence alignment (MSA) of sequence homologs in a protein family. A sequence profile is usually represented as a position-specific scoring matrix (PSSM) or an HMM (Hidden Markov Model) and accordingly PSSM-PSSM or HMM-HMM comparison is used for homolog detection. This paper presents a new homology detection method MRFalign, consisting of three key components: 1) a Markov Random Fields (MRF) representation of a protein family; 2) a scoring function measuring similarity of two MRFs; and 3) an efficient ADMM (Alternating Direction Method of Multipliers) algorithm aligning two MRFs. Compared to HMM that can only model very short-range residue correlation, MRFs can model long-range residue interaction pattern and thus, encode information for the global 3D structure of a protein family. Consequently, MRF-MRF comparison for remote homology detection shall be much more sensitive than HMM-HMM or PSSM-PSSM comparison. Experiments confirm that MRFalign outperforms several popular HMM or PSSM-based methods in terms of both alignment accuracy and remote homology detection and that MRFalign works particularly well for mainly beta proteins. For example, tested on the benchmark SCOP40 (8353 proteins) for homology detection, PSSM-PSSM and HMM-HMM succeed on 48% and 52% of proteins, respectively, at superfamily level, and on 15% and 27% of proteins, respectively, at fold level. In contrast, MRFalign succeeds on 57.3% and 42.5% of proteins at superfamily and fold level, respectively. This study implies that long-range residue interaction patterns are very helpful for sequence-based homology detection. The software is available for download at http://raptorx.uchicago.edu/download/. A summary of this paper appears in the proceedings of the RECOMB 2014 conference, April 2-5. PMID:24675572

  6. Butterfly eyespot serial homology: enter the Hox genes.

    PubMed

    Hombría, James Castelli-Gair

    2011-01-01

    Hox genes modify serial homology patterns in many organisms, exemplified in vertebrates by modification of the axial skeleton and in arthropods by diversification of the body segments. Butterfly wing eyespots also appear in a serial homologous pattern that, in certain species, is subject to local modification. A paper in EvoDevo reports the Hox gene Antp is the earliest known gene to have eyespot-specific expression; however, not all Lepidoptera express Antp in eyespots, suggesting some developmental flexibility. PMID:21527048

  7. Butterfly eyespot serial homology: enter the Hox genes.

    PubMed

    Hombría, James Castelli-Gair

    2011-01-01

    Hox genes modify serial homology patterns in many organisms, exemplified in vertebrates by modification of the axial skeleton and in arthropods by diversification of the body segments. Butterfly wing eyespots also appear in a serial homologous pattern that, in certain species, is subject to local modification. A paper in EvoDevo reports the Hox gene Antp is the earliest known gene to have eyespot-specific expression; however, not all Lepidoptera express Antp in eyespots, suggesting some developmental flexibility.

  8. Metagenomic gene annotation by a homology-independent approach

    SciTech Connect

    Froula, Jeff; Zhang, Tao; Salmeen, Annette; Hess, Matthias; Kerfeld, Cheryl A.; Wang, Zhong; Du, Changbin

    2011-06-02

    Fully understanding the genetic potential of a microbial community requires functional annotation of all the genes it encodes. The recently developed deep metagenome sequencing approach has enabled rapid identification of millions of genes from a complex microbial community without cultivation. Current homology-based gene annotation fails to detect distantly-related or structural homologs. Furthermore, homology searches with millions of genes are very computational intensive. To overcome these limitations, we developed rhModeller, a homology-independent software pipeline to efficiently annotate genes from metagenomic sequencing projects. Using cellulases and carbonic anhydrases as two independent test cases, we demonstrated that rhModeller is much faster than HMMER but with comparable accuracy, at 94.5percent and 99.9percent accuracy, respectively. More importantly, rhModeller has the ability to detect novel proteins that do not share significant homology to any known protein families. As {approx}50percent of the 2 million genes derived from the cow rumen metagenome failed to be annotated based on sequence homology, we tested whether rhModeller could be used to annotate these genes. Preliminary results suggest that rhModeller is robust in the presence of missense and frameshift mutations, two common errors in metagenomic genes. Applying the pipeline to the cow rumen genes identified 4,990 novel cellulases candidates and 8,196 novel carbonic anhydrase candidates.In summary, we expect rhModeller to dramatically increase the speed and quality of metagnomic gene annotation.

  9. Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum.

    PubMed Central

    Sauer, U; Treuner, A; Buchholz, M; Santangelo, J D; Dürre, P

    1994-01-01

    Using a PCR-based approach, we have cloned various sigma factor homologous genes from Clostridium acetobutylicum DSM 792. The nucleotide sequence of the dnaE-sigA operon has been determined and predicts two genes encoding 69- and 43-kDa proteins. The deduced DnaE amino acid sequence has approximately 30% amino acid identity with protein sequences of other primases. The putative sigA gene product shows high homology to primary sigma factors of various bacteria, most significantly to Bacillus subtilis and Staphylococcus aureus. Northern (RNA) blot analysis revealed that both genes from an operon, which is clearly expressed under conditions that allow for cell division. A promoter sequence with significant homology to the sigma H-dependent Bacillus promoters preceded the determined transcriptional start point, 182 bp upstream of the GUG start codon of dnaE. The homologous genes to Bacillus spp. sporulation sigma factors G, E, and K have been cloned and sequenced. Indirect evidence for the existence of sigma F was obtained by identification of a DNA sequence homologous to the respective Bacillus consensus promoter. Southern hybridization analysis indicated the presence of sigma D and sigma H homologous genes in C. acetobutylicum. A new gene group conserved within the eubacteria, but with yet unspecified functions, is described. The data presented here provide strong evidence that at least some of the complex regulation features of sporulation in B. subtilis are conserved in C. acetobutylicum and possibly Clostridium spp. Images PMID:7961408

  10. Involvement of homologous recombination repair after proton-induced DNA damage.

    PubMed

    Rostek, C; Turner, E L; Robbins, M; Rightnar, S; Xiao, W; Obenaus, A; Harkness, T A A

    2008-03-01

    Protection from chronic exposure to cosmic radiation, which is primarily composed of protons, in future manned missions to Mars and beyond is considered to be a key unresolved issue. To model the effects of cosmic radiation on a living cell, we used Saccharomyces cerevisiae cells harboring various deletions of DNA repair genes to investigate the response of cells to DNA strand breaks caused by exposure to 250 MeV proton irradiation (linear energy transfer of 0.41 keV/microm). In our study, DNA strand breaks induced by exposure to protons were predominantly repaired via the homologous recombination and postreplication repair pathways. We simulated chronic exposure to proton irradiation by treating cells from colonies that survived proton treatment, after several rounds of subculturing, to a second proton dose, as well as additional cell stressors. In general, cells cultured from proton surviving colonies were not more sensitive to secondary cell stressors. However, cells from rad52delta colonies that survived proton treatment showed increased resistance to secondary stressors, such as gamma-rays (1.17 and 1.33 MeV; 0.267 keV/microm), ultraviolet (UV) and proton irradiation and elevated temperatures. Resistance to secondary stressors was also observed in rad52delta cells that survived exposure to gamma-rays, rather than protons, but this was not observed to occur in rad52delta cells after UV irradiation. rad52delta cells that survived exposure to protons, followed by gamma-rays (proton surviving colonies were cultured prior to gamma-ray exposure), exhibited an additive effect, whereby these cells had a further increase in stress resistance. A genetic analysis indicated that increased stress resistance is most likely due to a second-site mutation that suppresses the rad52delta phenotype. We will discuss possible origins of these second-site mutations. PMID:18267950

  11. The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.

    PubMed Central

    Poon, D; Schroeder, S; Wang, C K; Yamamoto, T; Horikoshi, M; Roeder, R G; Weil, P A

    1991-01-01

    We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed. Images PMID:1922021

  12. The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis.

    PubMed Central

    Lin, F C; Arndt, K T

    1995-01-01

    We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102. At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis. These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis. Images PMID:7796803

  13. BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae

    PubMed Central

    1996-01-01

    The ellipsoidal shape of the yeast Saccharomyces cerevisiae is the result of successive isotropic/apical growth switches that are regulated in a cell cycle-dependent manner. It is thought that growth polarity is governed by the remodeling of the actin cytoskeleton that is itself under the control of the cell cycle machinery. The cell cycle and the morphogenesis cycle are tightly coupled and it has been recently suggested that a morphogenesis/polarity checkpoint control monitors bud emergence in order to maintain the coupling of these two events (Lew, D. J., and S. I. Reed. 1995. J. Cell Biol. 129:739-749). During a screen based on the inability of cells impaired in the budding process to survive when the morphogenesis checkpoint control is abolished, we identified and characterized BED1, a new gene that is required for efficient budding. Cells carrying a disrupted allele of BED1 no longer have the wild-type ellipsoidal shape characteristic of S. cerevisiae, are larger than wild-type cells, are deficient in bud emergence, and depend upon an intact morphogenesis checkpoint control to survive. These cells show defects in polarized growth despite the fact that the actin cytoskeleton appears normal. Our results suggest that Bed1 is a type II membrane protein localized in the endoplasmic reticulum. BED1 is significantly homologous to gma12+, a S. pombe gene coding for an alpha-1,2,-galactosyltransferase, suggesting that glycosylation of specific proteins or lipids could be important for signaling in the switch to polarized growth and in bud emergence. PMID:8567719

  14. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

    PubMed Central

    Tang, H Y; Cai, M

    1996-01-01

    Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae. PMID:8756649

  15. Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

    PubMed

    Gamo, F J; Lafuente, M J; Casamayor, A; Ariño, J; Aldea, M; Casas, C; Herrero, E; Gancedo, C

    1996-06-15

    We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

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

    PubMed

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

    2014-03-01

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

  17. Glucose- and nitrogen sensing and regulatory mechanisms in Saccharomyces cerevisiae.

    PubMed

    Rødkaer, Steven V; Faergeman, Nils J

    2014-08-01

    Pro- and eukaryotic cells are constantly challenged by varying concentrations of nutrients in their environment. Perceiving and adapting to such changes are therefore crucial for cellular viability. Thus, numerous specialized cellular receptors continuously sense and react to the availability of nutrients such as glucose and nitrogen. When stimulated, these receptors initiate various cellular signaling pathways, which in concert constitute a complex regulatory network. To ensure a highly specific response, these pathways and networks cross-communicate with each other and are regulated at several steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae.

  18. Membrane Protein Production in the Yeast, S. cerevisiae.

    PubMed

    Cartwright, Stephanie P; Mikaliunaite, Lina; Bill, Roslyn M

    2016-01-01

    The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes. PMID:27485327

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

    PubMed

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

    2016-06-01

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

  20. Isolation of an osmotolerant ale strain of Saccharomyces cerevisiae.

    PubMed

    Pironcheva, G

    1998-01-01

    Saccharomyces cerevisiae (ale strain) grown in batch culture to stationary phase was tested for its tolerance to heat (50 degrees C for 5 min), hydrogen peroxide (0.3 M) and salt (growth in 1.5 M sodium chloride/YPD medium). Yeast cells which have been exposed previously to heat shock are more tolerant to hydrogen peroxide and high salt concentrations (1.5 M NaCl) than the controls. Their fermentative activity as judged by glucose consumption and their viability, as judged by cell number and density have higher levels when compared with cells not previously exposed to heat shock. Experimental conditions facilitated the isolation of S. cerevisiae ale strain, which was tolerant to heat, and other agents such as hydrogen peroxide and sodium chloride.

  1. Using weighted features to predict recombination hotspots in Saccharomyces cerevisiae.

    PubMed

    Liu, Guoqing; Xing, Yongqiang; Cai, Lu

    2015-10-01

    Characterization and accurate prediction of recombination hotspots and coldspots have crucial implications for the mechanism of recombination. Several models have predicted recombination hot/cold spots successfully, but there is still much room for improvement. We present a novel classifier in which k-mer frequency, physical and thermodynamic properties of DNA sequences are incorporated in the form of weighted features. Applying the classifier to recombination hot/cold ORFs in Saccharomyces cerevisiae, we achieved an accuracy of 90%, which is ~5% higher than existing methods, such as iRSpot-PseDNC, IDQD and Random Forest. The model also predicted non-ORF recombination hot/cold spots sequences in S. cerevisiae with high accuracy. A broad applicability of the model in the field of classification is expected.

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

    PubMed Central

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

    1999-01-01

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

  3. Advanced biofuel production by the yeast Saccharomyces cerevisiae.

    PubMed

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

    2013-06-01

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

  4. Purification of fluorescently labeled Saccharomyces cerevisiae Spindle Pole Bodies

    PubMed Central

    Davis, Trisha N.

    2016-01-01

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

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

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

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

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

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

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

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

    SciTech Connect

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

    1986-04-01

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

  8. ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

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

    2015-01-01

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

  9. Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae.

    PubMed

    Young, M; Davies, M J; Bailey, D; Gradwell, M J; Smestad-Paulsen, B; Wold, J K; Barnes, R M; Hounsell, E F

    1998-08-01

    Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manalpha1-->3Manalpha1-->2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.

  10. Transformation of Mucor circinelloides with autoreplicative vectors containing homologous and heterologous ARS elements and the dominant Cbx(r) carboxine-resistance gene.

    PubMed

    Ortiz-Alvarado, R; Gonzalez-Hernandez, G A; Torres-Guzman, J C; Gutierrez-Corona, J F

    2006-03-01

    Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu(+) Cbx(r)) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx(r)(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 mu plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65-120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.

  11. Saccharomyces cerevisiae engineered to produce D-xylonate.

    PubMed

    Toivari, Mervi H; Ruohonen, Laura; Richard, Peter; Penttilä, Merja; Wiebe, Marilyn G

    2010-10-01

    Saccharomyces cerevisiae was engineered to produce D-xylonate by introducing the Trichoderma reesei xyd1 gene, encoding a D-xylose dehydrogenase. D-xylonate was not toxic to S. cerevisiae, and the cells were able to export D-xylonate produced in the cytoplasm to the supernatant. Up to 3.8 g of D-xylonate per litre, at rates of 25-36 mg of D-xylonate per litre per hour, was produced. Up to 4.8 g of xylitol per litre was also produced. The yield of D-xylonate from D-xylose was approximately 0.4 g of D-xylonate per gramme of D-xylose consumed. Deletion of the aldose reductase encoding gene GRE3 in S. cerevisiae strains expressing xyd1 reduced xylitol production by 67%, increasing the yield of D-xylonate from D-xylose. However, D-xylose uptake was reduced compared to strains containing GRE3, and the total amount of D-xylonate produced was reduced. To determine whether the co-factor NADP+ was limiting for D-xylonate production the Escherichia coli transhydrogenase encoded by udhA, the Bacillus subtilis glyceraldehyde 3-phosphate dehydrogenase encoded by gapB or the S. cerevisiae glutamate dehydrogenase encoded by GDH2 was co-expressed with xyd1 in the parent and GRE3 deficient strains. Although each of these enzymes enhanced NADPH consumption on D-glucose, they did not enhance D-xylonate production, suggesting that NADP+ was not the main limitation in the current D-xylonate producing strains.

  12. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants.

    PubMed

    Zhou, G L; Miyazaki, Y; Nakagawa, T; Tanaka, K; Shishido, K; Matsuda, H; Kawamukai, M

    1998-04-01

    The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function. PMID:9579081

  13. Overproduction of fatty acids in engineered Saccharomyces cerevisiae.

    PubMed

    Li, Xiaowei; Guo, Daoyi; Cheng, Yongbo; Zhu, Fayin; Deng, Zixin; Liu, Tiangang

    2014-09-01

    The long hydrocarbon fatty acyl chain is energy rich, making it an ideal precursor for liquid transportation fuels and high-value oleo chemicals. As Saccharomyces cerevisiae has many advantages for industrial production compared to Escherichia coli. Here, we attempted to engineer Saccharomyces cerevisiae for overproduction of fatty acids. First, disruption of the beta-oxidation pathway, elimination of the acyl-CoA synthetases, overexpression of different thioesterases and acetyl-CoA carboxylase ACC1, and engineering the supply of precursor acetyl-CoA. The engineered strain XL122 produced more than 120 mg/L of fatty acids. In parallel, we inactivated ADH1, the dominant gene for ethanol production, to redirect the metabolic flux to fatty acids synthesis. The engineered strain DG005 produced about 140 mg/L fatty acids. Additionally, Acetyl-CoA carboxylase was identified as a critical bottleneck of fatty acids synthesis in S. cerevisiae with a cell-free system. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 were further identified. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step. All these efforts, particularly the discovery of the limiting step are critical for developing a "cell factory" for the overproduction of fatty acids by using type I fatty acids synthase in yeast or other fungi. PMID:24752690

  14. Can Yeast (S. cerevisiae) Metabolic Volatiles Provide Polymorphic Signaling?

    PubMed Central

    Arguello, J. Roman; Sellanes, Carolina; Lou, Yann Ru; Raguso, Robert A.

    2013-01-01

    Chemical signaling between organisms is a ubiquitous and evolutionarily dynamic process that helps to ensure mate recognition, location of nutrients, avoidance of toxins, and social cooperation. Evolutionary changes in chemical communication systems progress through natural variation within the organism generating the signal as well as the responding individuals. A promising yet poorly understood system with which to probe the importance of this variation exists between D. melanogaster and S. cerevisiae. D. melanogaster relies on yeast for nutrients, while also serving as a vector for yeast cell dispersal. Both are outstanding genetic and genomic models, with Drosophila also serving as a preeminent model for sensory neurobiology. To help develop these two genetic models as an ecological model, we have tested if - and to what extent - S. cerevisiae is capable of producing polymorphic signaling through variation in metabolic volatiles. We have carried out a chemical phenotyping experiment for 14 diverse accessions within a common garden random block design. Leveraging genomic sequences for 11 of the accessions, we ensured a genetically broad sample and tested for phylogenetic signal arising from phenotypic dataset. Our results demonstrate that significant quantitative differences for volatile blends do exist among S. cerevisiae accessions. Of particular ecological relevance, the compounds driving the blend differences (acetoin, 2-phenyl ethanol and 3-methyl-1-butanol) are known ligands for D. melanogasters chemosensory receptors, and are related to sensory behaviors. Though unable to correlate the genetic and volatile measurements, our data point clear ways forward for behavioral assays aimed at understanding the implications of this variation. PMID:23990899

  15. Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter.

    PubMed

    Aliverdieva, D A; Mamaev, D V; Bondarenko, D I; Sholtz, K F

    2006-10-01

    Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).

  16. The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  17. PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. Results In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. Conclusion PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity. PMID:20507616

  18. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

    PubMed

    Vemuri, G N; Eiteman, M A; McEwen, J E; Olsson, L; Nielsen, J

    2007-02-13

    Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

  19. Membrane trafficking in the yeast Saccharomyces cerevisiae model.

    PubMed

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

    2015-01-09

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  2. Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae.

    PubMed

    Meca, G; Zhou, T; Li, X Z; Ritieni, A; Mañes, J

    2013-09-01

    The interaction between the mycotoxin beauvericin (BEA) and 9 yeast strains of Saccharomyces cerevisiae named LO9, YE-2, YE5, YE-6, YE-4, A34, A17, A42 and A08 was studied. The biological degradations were carried out under aerobic conditions in the liquid medium of Potato Dextrose Broth (PDB) at 25°C for 48 h and in a food/feed system composed of corn flour at 37°C for 3 days, respectively. BEA present in fermented medium and corn flour was determined using liquid chromatography coupled to the mass spectrometry detector in tandem (LC-MS/MS) and the BEA degradation products produced during the fermentations were determined using the technique of the liquid chromatography coupled to a linear ion trap (LIT). Results showed that the S. cerevisiae strains reduced meanly the concentration of the BEA present in PDB by 86.2% and in a food system by 71.1%. All the S. cerevisiae strains used in this study showed a significant BEA reduction during the fermentation process employed.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Role of social wasps in Saccharomyces cerevisiae ecology and evolution.

    PubMed

    Stefanini, Irene; Dapporto, Leonardo; Legras, Jean-Luc; Calabretta, Antonio; Di Paola, Monica; De Filippo, Carlotta; Viola, Roberto; Capretti, Paolo; Polsinelli, Mario; Turillazzi, Stefano; Cavalieri, Duccio

    2012-08-14

    Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain's evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals.

  7. Cell density-dependent linoleic acid toxicity to Saccharomyces cerevisiae.

    PubMed

    Ferreira, Túlio César; de Moraes, Lídia Maria Pepe; Campos, Elida Geralda

    2011-08-01

    Since the discovery of the apoptotic pathway in Saccharomyces cerevisiae, several compounds have been shown to cause apoptosis in this organism. While the toxicity of polyunsaturated fatty acids (PUFA) peroxides towards S. cerevisiae has been known for a long time, studies on the effect of nonoxidized PUFA are scarce. The present study deals specifically with linoleic acid (LA) in its nonoxidized form and investigates its toxicity to yeast. Saccharomyces cerevisiae is unable to synthesize PUFA, but can take up and incorporate them into its membranes. Reports from the literature indicate that LA is not toxic to yeast cells. However, we demonstrated that yeast cell growth decreased in cultures treated with 0.1 mM LA for 4 h, and 3-(4,5 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (a measure of respiratory activity) decreased by 47%. This toxicity was dependent on the number of cells used in the experiment. We show apoptosis induction by LA concomitant with increases in malondialdehyde, glutathione content, activities of catalase and cytochrome c peroxidase, and decreases in two metabolic enzyme activities. While the main purpose of this study was to show that LA causes cell death in yeast, our results indicate some of the molecular mechanisms of the cell toxicity of PUFA. PMID:21457450

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

    PubMed

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

    2015-09-01

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

  9. Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae

    PubMed Central

    Belyy, Alexander; Levanova, Nadezhda; Tabakova, Irina; Rospert, Sabine

    2016-01-01

    ABSTRACT The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5′ untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62–K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single

  10. Surfeit locus gene homologs are widely distributed in invertebrate genomes.

    PubMed

    Armes, N; Fried, M

    1996-10-01

    The mouse Surfeit locus contains six sequence-unrelated genes (Surf-1 to -6) arranged in the tightest gene cluster so far described for mammals. The organization and juxtaposition of five of the Surfeit genes (Surf-1 to -5) are conserved between mammals and birds, and this may reflect a functional or regulatory requirement for the gene clustering. We have undertaken an evolutionary study to determine whether the Surfeit genes are conserved and clustered in invertebrate genomes. Drosophila melanogaster and Caenorhabditis elegans homologs of the mouse Surf-4 gene, which encodes an integral membrane protein associated with the endoplasmic reticulum, have been isolated. The amino acid sequences of the Drosophila and C. elegans homologs are highly conserved in comparison with the mouse Surf-4 protein. In particular, a dilysine motif implicated in endoplasmic reticulum localization of the mouse protein is conserved in the invertebrate homologs. We show that the Drosophila Surf-4 gene, which is transcribed from a TATA-less promoter, is not closely associated with other Drosophila Surfeit gene homologs but rather is located upstream from sequences encoding a homolog of a yeast seryl-tRNA synthetase protein. There are at least two closely linked Surf-3/rpL7a genes or highly polymorphic alleles of a single Surf-3/rpL7a gene in the C. elegans genome. The chromosomal locations of the C. elegans Surf-1, Surf-3/rpL7a, and Surf-4 genes have been determined. In D. melanogaster the Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs and in C. elegans the Surf-1, Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs are located on completely different chromosomes, suggesting that any requirement for the tight clustering of the genes in the Surfeit locus is restricted to vertebrate lineages.

  11. Homologous prominence non-radial eruptions: A case study

    NASA Astrophysics Data System (ADS)

    Duchlev, P.; Koleva, K.; Madjarska, M. S.; Dechev, M.

    2016-10-01

    The present study provides important details on homologous eruptions of a solar prominence that occurred in active region NOAA 10904 on 2006 August 22. We report on the pre-eruptive phase of the homologous feature as well as the kinematics and the morphology of a forth from a series of prominence eruptions that is critical in defining the nature of the previous consecutive eruptions. The evolution of the overlying coronal field during homologous eruptions is discussed and a new observational criterion for homologous eruptions is provided. We find a distinctive sequence of three activation periods each of them containing pre-eruptive precursors such as a brightening and enlarging of the prominence body followed by small surge-like ejections from its southern end observed in the radio 17 GHz. We analyse a fourth eruption that clearly indicates a full reformation of the prominence after the third eruption. The fourth eruption although occurring 11 h later has an identical morphology, the same angle of propagation with respect to the radial direction, as well as similar kinematic evolution as the previous three eruptions. We find an important feature of the homologous eruptive prominence sequence that is the maximum height increase of each consecutive eruption. The present analysis establishes that all four eruptions observed in Hα are of confined type with the third eruption undergoing a thermal disappearance during its eruptive phase. We suggest that the observation of the same direction of the magnetic flux rope (MFR) ejections can be consider as an additional observational criterion for MFR homology. This observational indication for homologous eruptions is important, especially in the case of events of typical or poorly distinguishable morphology of eruptive solar phenomena.

  12. FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter.

    PubMed

    Galeote, Virginie; Novo, Maïté; Salema-Oom, Madalena; Brion, Christian; Valério, Elisabete; Gonçalves, Paula; Dequin, Sylvie

    2010-12-01

    Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

  13. Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5'- to 3'-Helicase Activity with a Preference for Translocation on DNA Rather than RNA.

    PubMed

    Martin-Tumasz, Stephen; Brow, David A

    2015-09-18

    In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as a recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here, we report the purification and characterization of the 89-kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops.

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

    PubMed

    Park, E; Guzder, S N; Koken, M H; Jaspers-Dekker, I; Weeda, G; Hoeijmakers, J H; Prakash, S; Prakash, L

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M(r) 95,356). The RAD25 (SSL2)- and XPBC-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UV sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability. PMID:1333609

  15. A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis.

    PubMed

    Hanada, K; Hara, T; Nishijima, M; Kuge, O; Dickson, R C; Nagiec, M M

    1997-12-19

    Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT. PMID:9405408

  16. Recombination-Mediated Telomere Maintenance in Saccharomyces cerevisiae Is Not Dependent on the Shu Complex.

    PubMed

    van Mourik, Paula M; de Jong, Jannie; Agpalo, Danielle; Claussin, Clémence; Rothstein, Rodney; Chang, Michael

    2016-01-01

    In cells lacking telomerase, telomeres shorten progressively during each cell division due to incomplete end-replication. When the telomeres become very short, cells enter a state that blocks cell division, termed senescence. A subset of these cells can overcome senescence and maintain their telomeres using telomerase-independent mechanisms. In Saccharomyces cerevisiae, these cells are called 'survivors' and are dependent on Rad52-dependent homologous recombination and Pol32-dependent break-induced replication. There are two main types of survivors: type I and type II. The type I survivors require Rad51 and maintain telomeres by amplification of subtelomeric elements, while the type II survivors are Rad51-independent, but require the MRX complex and Sgs1 to amplify the C1-3A/TG1-3 telomeric sequences. Rad52, Pol32, Rad51, and Sgs1 are also important to prevent accelerated senescence, indicating that recombination processes are important at telomeres even before the formation of survivors. The Shu complex, which consists of Shu1, Shu2, Psy3, and Csm2, promotes Rad51-dependent homologous recombination and has been suggested to be important for break-induced replication. It also promotes the formation of recombination intermediates that are processed by the Sgs1-Top3-Rmi1 complex, as mutations in the SHU genes can suppress various sgs1, top3, and rmi1 mutant phenotypes. Given the importance of recombination processes during senescence and survivor formation, and the involvement of the Shu complex in many of the same processes during DNA repair, we hypothesized that the Shu complex may also have functions at telomeres. Surprisingly, we find that this is not the case: the Shu complex does not affect the rate of senescence, does not influence survivor formation, and deletion of SHU1 does not suppress the rapid senescence and type II survivor formation defect of a telomerase-negative sgs1 mutant. Altogether, our data suggest that the Shu complex is not important for

  17. Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.

    PubMed

    Tishkoff, D X; Johnson, A W; Kolodner, R D

    1991-05-01

    Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is

  18. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

    PubMed Central

    Siddiqui, A H; Brandriss, M C

    1989-01-01

    The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction. Images PMID:2689862

  19. Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair.

    PubMed Central

    Verhage, R A; van Gool, A J; de Groot, N; Hoeijmakers, J H; van de Putte, P; Brouwer, J

    1996-01-01

    The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26. PMID:8552076

  20. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

    PubMed Central

    Argueso, Juan Lucas; Carazzolle, Marcelo F.; Mieczkowski, Piotr A.; Duarte, Fabiana M.; Netto, Osmar V.C.; Missawa, Silvia K.; Galzerani, Felipe; Costa, Gustavo G.L.; Vidal, Ramon O.; Noronha, Melline F.; Dominska, Margaret; Andrietta, Maria G.S.; Andrietta, Sílvio R.; Cunha, Anderson F.; Gomes, Luiz H.; Tavares, Flavio C.A.; Alcarde, André R.; Dietrich, Fred S.; McCusker, John H.; Petes, Thomas D.; Pereira, Gonçalo A.G.

    2009-01-01

    Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (∼2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies. PMID:19812109

  1. A complete set of marked telomeres in Saccharomyces cerevisiae for physical mapping and cloning

    SciTech Connect

    Louis, E.J.; Borts, R.H.

    1995-01-01

    Each telomere in a single strain (S288C) of Saccharomyces cerevisiae was marked with a URA3 containing integrating vector having telomeric TG{sub 1-3} sequences. Efficiency of integrative transformation was enhanced by creating single random double-strand breaks in the integrating vector using DNAseI in the presence of Mn{sup 2+} ions. A total of 327 transformants were screened by CHEF gels of intact chromosomal DNA. Transformants with homology to the vector at particular chromosomal bands were then screened by Southern analysis with several restriction enzymes to confirm telomeric locations. CHEF gels of NotI and/or SfiI digests were also analyzed to determine left or right arm locations. In some cases allelism of marked telomeres was determined genetically. Transformation was performed by lithium acetate and electroporation with varying results. Electroporation resulted in 50% (75/150) of the integrants at the internal URA3 location rather than telomeres. There were also two rearrangements involving URA3 and the telomere of another chromosome. Lithium acetate transformation resulted in fewer integrants at the internal URA3 location (5/84) and no rearrangements. All telomeres were marked with approximately the same efficiency ranging from 0 to 11 hits in the first 240 transformants. These marked telomeres can be used to complete the physical maps of chromosomes in which the telomere regions are absent. The marked telomeres can be cloned with the appropriate restriction enzymes, thus completing the cloning of individual chromosomes for sequencing projects. The analysis of these clones will lead to a better understanding of telomere region biology. The methodology can also be applied to telomeres of other organisms once they are cloned as telomeric YACs. 36 refs., 7 figs., 2 tabs.

  2. A Genome-Wide Screen with Nicotinamide to Identify Sirtuin-Dependent Pathways in Saccharomyces cerevisiae

    PubMed Central

    Choy, John S.; Qadri, Bayan; Henry, Leah; Shroff, Kunal; Bifarin, Olatomiwa; Basrai, Munira A.

    2015-01-01

    Sirtuins are evolutionarily conserved NAD-dependent deacetylases that catalyze the cleavage of NAD+ into nicotinamide (NAM), which can act as a pan-sirtuin inhibitor in unicellular and multicellular organisms. Sirtuins regulate processes such as transcription, DNA damage repair, chromosome segregation, and longevity extension in yeast and metazoans. The founding member of the evolutionarily conserved sirtuin family, SIR2, was first identified in budding yeast. Subsequent studies led to the identification of four yeast SIR2 homologs HST1, HST2, HST3, and HST4. Understanding the downstream physiological consequences of inhibiting sirtuins can be challenging since most studies focus on single or double deletions of sirtuins, and mating defects in SIR2 deletions hamper genome-wide screens. This represents an important gap in our knowledge of how sirtuins function in highly complex biological processes such as aging, metabolism, and chromosome segregation. In this report, we used a genome-wide screen to explore sirtuin-dependent processes in Saccharomyces cerevisiae by identifying deletion mutants that are sensitive to NAM. We identified 55 genes in total, 36 of which have not been previously reported to be dependent on sirtuins. We find that genome stability pathways are particularly vulnerable to loss of sirtuin activity. Here, we provide evidence that defects in sister chromatid cohesion renders cells sensitive to growth in the presence of NAM. The results of our screen provide a broad view of the biological pathways sensitive to inhibition of sirtuins, and advance our understanding of the function of sirtuins and NAD+ biology. PMID:26646153

  3. A Genome-Wide Screen with Nicotinamide to Identify Sirtuin-Dependent Pathways in Saccharomyces cerevisiae.

    PubMed

    Choy, John S; Qadri, Bayan; Henry, Leah; Shroff, Kunal; Bifarin, Olatomiwa; Basrai, Munira A

    2016-02-01

    Sirtuins are evolutionarily conserved NAD-dependent deacetylases that catalyze the cleavage of NAD(+) into nicotinamide (NAM), which can act as a pan-sirtuin inhibitor in unicellular and multicellular organisms. Sirtuins regulate processes such as transcription, DNA damage repair, chromosome segregation, and longevity extension in yeast and metazoans. The founding member of the evolutionarily conserved sirtuin family, SIR2, was first identified in budding yeast. Subsequent studies led to the identification of four yeast SIR2 homologs HST1, HST2, HST3, and HST4. Understanding the downstream physiological consequences of inhibiting sirtuins can be challenging since most studies focus on single or double deletions of sirtuins, and mating defects in SIR2 deletions hamper genome-wide screens. This represents an important gap in our knowledge of how sirtuins function in highly complex biological processes such as aging, metabolism, and chromosome segregation. In this report, we used a genome-wide screen to explore sirtuin-dependent processes in Saccharomyces cerevisiae by identifying deletion mutants that are sensitive to NAM. We identified 55 genes in total, 36 of which have not been previously reported to be dependent on sirtuins. We find that genome stability pathways are particularly vulnerable to loss of sirtuin activity. Here, we provide evidence that defects in sister chromatid cohesion renders cells sensitive to growth in the presence of NAM. The results of our screen provide a broad view of the biological pathways sensitive to inhibition of sirtuins, and advance our understanding of the function of sirtuins and NAD(+) biology. PMID:26646153

  4. Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose.

    PubMed Central

    Gasent-Ramírez, J M; Codón, A C; Benítez, T

    1995-01-01

    Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7793932

  5. Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

    PubMed Central

    Zanchin, N I; Roberts, P; DeSilva, A; Sherman, F; Goldfarb, D S

    1997-01-01

    The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells. PMID:9271378

  6. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production.

    PubMed

    Argueso, Juan Lucas; Carazzolle, Marcelo F; Mieczkowski, Piotr A; Duarte, Fabiana M; Netto, Osmar V C; Missawa, Silvia K; Galzerani, Felipe; Costa, Gustavo G L; Vidal, Ramon O; Noronha, Melline F; Dominska, Margaret; Andrietta, Maria G S; Andrietta, Sílvio R; Cunha, Anderson F; Gomes, Luiz H; Tavares, Flavio C A; Alcarde, André R; Dietrich, Fred S; McCusker, John H; Petes, Thomas D; Pereira, Gonçalo A G

    2009-12-01

    Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

  7. Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae.

    PubMed Central

    Kornuc, M; Altman, R; Harrich, D; Garcia, J; Chao, J; Kayne, P; Gaynor, R

    1988-01-01

    The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells. Images PMID:2975753

  8. Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae.

    PubMed

    Kornuc, M; Altman, R; Harrich, D; Garcia, J; Chao, J; Kayne, P; Gaynor, R

    1988-09-01

    The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

  9. The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, P R; Biggar, S; Prakash, L; Prakash, S

    1992-01-01

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities. Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe. RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids. We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair. We find that the RAD3 and rhp3+ genes can functionally substitute for one another. The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels. These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions. Images PMID:1534406

  10. The NAM2 proteins from S. cerevisiae and S. douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing.

    PubMed Central

    Herbert, C J; Labouesse, M; Dujardin, G; Slonimski, P P

    1988-01-01

    We have cloned and sequenced the NAM2 gene of Saccharomyces douglasii, which is a homologue of the NAM2 gene of Saccharomyces cerevisiae. The wild-type S.douglasii gene possesses the suppressor functions of the mutant S. cerevisiae NAM2-1 allele, being able to cure a mitochondrial b14 maturase deficiency. By sequence comparisons and direct measurements we have demonstrated that the NAM2 genes encode mitochondrial leucyl tRNA synthetases (EC 6.1.1.4.). Using a derivative of the NAM2 gene, where the expression of the gene is under the control of the UAS GAL10, we have shown that the processing of the pre-mRNA from the two mosaic genes oxi3 and cob-box is impaired when transcription of the gene is repressed. These results lead us to conclude that the mitochondrial leucyl tRNA synthetase is involved in protein synthesis and mRNA splicing. Sequence comparisons show that the mitochondrial and Escherichia coli leucyl tRNA synthetases are highly homologous; however, significant features which may be important for the splicing functions of the mitochondrial enzymes are absent from the bacterial enzyme. Images PMID:3284745

  11. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    PubMed Central

    Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

    2008-01-01

    Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose

  12. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  13. Multiscale analysis of nonlinear systems using computational homology

    SciTech Connect

    Konstantin Mischaikow, Rutgers University /Georgia Institute of Technology, Michael Schatz, Georgia Institute of Technology, William Kalies, Florida Atlantic University, Thomas Wanner,George Mason University

    2010-05-19

    This is a collaborative project between the principal investigators. However, as is to be expected, different PIs have greater focus on different aspects of the project. This report lists these major directions of research which were pursued during the funding period: (1) Computational Homology in Fluids - For the computational homology effort in thermal convection, the focus of the work during the first two years of the funding period included: (1) A clear demonstration that homology can sensitively detect the presence or absence of an important flow symmetry, (2) An investigation of homology as a probe for flow dynamics, and (3) The construction of a new convection apparatus for probing the effects of large-aspect-ratio. (2) Computational Homology in Cardiac Dynamics - We have initiated an effort to test the use of homology in characterizing data from both laboratory experiments and numerical simulations of arrhythmia in the heart. Recently, the use of high speed, high sensitivity digital imaging in conjunction with voltage sensitive fluorescent dyes has enabled researchers to visualize electrical activity on the surface of cardiac tissue, both in vitro and in vivo. (3) Magnetohydrodynamics - A new research direction is to use computational homology to analyze results of large scale simulations of 2D turbulence in the presence of magnetic fields. Such simulations are relevant to the dynamics of black hole accretion disks. The complex flow patterns from simulations exhibit strong qualitative changes as a function of magnetic field strength. Efforts to characterize the pattern changes using Fourier methods and wavelet analysis have been unsuccessful. (4) Granular Flow - two experts in the area of granular media are studying 2D model experiments of earthquake dynamics where the stress fields can be measured; these stress fields from complex patterns of 'force chains' that may be amenable to analysis using computational homology. (5) Microstructure Characterization

  14. Multiscale analysis of nonlinear systems using computational homology

    SciTech Connect

    Konstantin Mischaikow; Michael Schatz; William Kalies; Thomas Wanner

    2010-05-24

    This is a collaborative project between the principal investigators. However, as is to be expected, different PIs have greater focus on different aspects of the project. This report lists these major directions of research which were pursued during the funding period: (1) Computational Homology in Fluids - For the computational homology effort in thermal convection, the focus of the work during the first two years of the funding period included: (1) A clear demonstration that homology can sensitively detect the presence or absence of an important flow symmetry, (2) An investigation of homology as a probe for flow dynamics, and (3) The construction of a new convection apparatus for probing the effects of large-aspect-ratio. (2) Computational Homology in Cardiac Dynamics - We have initiated an effort to test the use of homology in characterizing data from both laboratory experiments and numerical simulations of arrhythmia in the heart. Recently, the use of high speed, high sensitivity digital imaging in conjunction with voltage sensitive fluorescent dyes has enabled researchers to visualize electrical activity on the surface of cardiac tissue, both in vitro and in vivo. (3) Magnetohydrodynamics - A new research direction is to use computational homology to analyze results of large scale simulations of 2D turbulence in the presence of magnetic fields. Such simulations are relevant to the dynamics of black hole accretion disks. The complex flow patterns from simulations exhibit strong qualitative changes as a function of magnetic field strength. Efforts to characterize the pattern changes using Fourier methods and wavelet analysis have been unsuccessful. (4) Granular Flow - two experts in the area of granular media are studying 2D model experiments of earthquake dynamics where the stress fields can be measured; these stress fields from complex patterns of 'force chains' that may be amenable to analysis using computational homology. (5) Microstructure Characterization

  15. Tocopherol and tocotrienol homologs in parenteral lipid emulsions

    PubMed Central

    Xu, Zhidong; Harvey, Kevin A; Pavlina, Thomas M; Zaloga, Gary P; Siddiqui, Rafat A

    2015-01-01

    Parenteral lipid emulsions, which are made of oils from plant and fish sources, contain different types of tocopherols and tocotrienols (vitamin E homologs). The amount and types of vitamin E homologs in various lipid emulsions vary considerably and are not completely known. The objective of this analysis was to develop a quantitative method to determine levels of all vitamin E homologs in various lipid emulsions. An HPLC system was used to measure vitamin E homologs using a Pinnacle DB Silica normal phase column and an isocratic, n-hexane:1,4 dioxane (98:2) mobile phase. An optimized protocol was used to report vitamin E homolog concentrations in soybean oil-based (Intralipid®, Ivelip®, Lipofundin® N, Liposyn® III, and Liposyn® II), medium- and long-chain fatty acid-based (Lipofundin®, MCT and Structolipid®), olive oil-based (ClinOleic®), and fish oil-based (Omegaven®) and mixture of these oils-based (SMOFlipid®, Lipidem®) commercial parenteral lipid emulsions. Total content of all vitamin E homologs varied greatly between different emulsions, ranging from 57.9 to 383.9 µg/mL. Tocopherols (α, β, γ, δ) were the predominant vitamin E homologs for all emulsions, with tocotrienol content < 0.3%. In all of the soybean emulsions, except for Lipofundin® N, the predominant vitamin E homolog was γ-tocopherol, which ranged from 57–156 µg/mL. ClinOleic® predominantly contained α-tocopherol (32 µg/mL), whereas α-tocopherol content in Omegaven® was higher than most of the other lipid emulsions (230 µg/mL). Practical applications The information on the types and quantity of vitamin E homologs in various lipid emulsions will be extremely useful to physicians and healthcare personnel in selecting appropriate lipid emulsions that are exclusively used in patients with inadequate gastrointestinal function, including hospitalized and critically ill patients. Some emulsions may require vitamin E supplementation in order to meet minimal human requirements

  16. DNA sequence alignment by microhomology sampling during homologous recombination

    PubMed Central

    Qi, Zhi; Redding, Sy; Lee, Ja Yil; Gibb, Bryan; Kwon, YoungHo; Niu, Hengyao; Gaines, William A.; Sung, Patrick

    2015-01-01

    Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination. PMID:25684365

  17. Homology modeling a fast tool for drug discovery: current perspectives.

    PubMed

    Vyas, V K; Ukawala, R D; Ghate, M; Chintha, C

    2012-01-01

    Major goal of structural biology involve formation of protein-ligand complexes; in which the protein molecules act energetically in the course of binding. Therefore, perceptive of protein-ligand interaction will be very important for structure based drug design. Lack of knowledge of 3D structures has hindered efforts to understand the binding specificities of ligands with protein. With increasing in modeling software and the growing number of known protein structures, homology modeling is rapidly becoming the method of choice for obtaining 3D coordinates of proteins. Homology modeling is a representation of the similarity of environmental residues at topologically corresponding positions in the reference proteins. In the absence of experimental data, model building on the basis of a known 3D structure of a homologous protein is at present the only reliable method to obtain the structural information. Knowledge of the 3D structures of proteins provides invaluable insights into the molecular basis of their functions. The recent advances in homology modeling, particularly in detecting and aligning sequences with template structures, distant homologues, modeling of loops and side chains as well as detecting errors in a model contributed to consistent prediction of protein structure, which was not possible even several years ago. This review focused on the features and a role of homology modeling in predicting protein structure and described current developments in this field with victorious applications at the different stages of the drug design and discovery.

  18. Sorption of linear alcohol ethoxylate surfactant homologs to soils

    NASA Astrophysics Data System (ADS)

    Yuan, Ching; Jafvert, Chad T.

    1997-11-01

    Sorption onto five saturated soils of the homologs within the commercial surfactant mixture Brij 35 (registered trademark of ICI Americas) was investigated. Brij 35 is a mixture of linear ethoxylated alcohols, having an average of 23 ethoxy (EO) groups per molecule and alcohol chain of primarily 12 carbons in length (C 12H 25(OCH 2CH 2) 23OH). In experiments, saturated soils were exposed to various concentrations of the surfactant mixture for specified times, the slurries were centrifuged to separate the phases, the aqueous phases were extracted with 1,2-dichloroethane, and the residual homologs were derivatized with 3,5-dinitrobenzoyl chloride and analyzed by normal phase HPLC. Homologs containing 4-43 EO groups were chromatographically separated at near baseline. At aqueous Brij 35 concentrations below the critical micelle concentration (cmc), the proportion of each homolog sorbed to each of the soils increased with increasing EO chain length through the homologous series. As a result, in experiments where a significant proportion of the surfactant adsorbed, significant shifts in the aqueous phase compositions occurred to mixtures with lower mean EO numbers. A sharp break in the adsorption isotherms occurs at the cmc.

  19. Primary homologies of the circumorbital bones of snakes.

    PubMed

    Palci, Alessandro; Caldwell, Michael W

    2013-09-01

    Some snakes have two circumorbital ossifications that in the current literature are usually referred to as the postorbital and supraorbital. We review the arguments that have been proposed to justify this interpretation and provide counter-arguments that reject those conjectures of primary homology based on the observation of 32 species of lizards and 81 species of snakes (both extant and fossil). We present similarity arguments, both topological and structural, for reinterpretation of the primary homologies of the dorsal and posterior orbital ossifications of snakes. Applying the test of similarity, we conclude that the posterior orbital ossification of snakes is topologically consistent as the homolog of the lacertilian jugal, and that the dorsal orbital ossification present in some snakes (e.g., pythons, Loxocemus, and Calabaria) is the homolog of the lacertilian postfrontal. We therefore propose that the terms postorbital and supraorbital should be abandoned as reference language for the circumorbital bones of snakes, and be replaced with the terms jugal and postfrontal, respectively. The primary homology claim for the snake "postorbital" fails the test of similarity, while the term "supraorbital" is an unnecessary and inaccurate application of the concept of a neomorphic ossification, for an element that passes the test of similarity as a postfrontal. This reinterpretation of the circumorbital bones of snakes is bound to have important repercussions for future phylogenetic analyses and consequently for our understanding of the origin and evolution of snakes.

  20. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  1. Primary homologies of the circumorbital bones of snakes.

    PubMed

    Palci, Alessandro; Caldwell, Michael W

    2013-09-01

    Some snakes have two circumorbital ossifications that in the current literature are usually referred to as the postorbital and supraorbital. We review the arguments that have been proposed to justify this interpretation and provide counter-arguments that reject those conjectures of primary homology based on the observation of 32 species of lizards and 81 species of snakes (both extant and fossil). We present similarity arguments, both topological and structural, for reinterpretation of the primary homologies of the dorsal and posterior orbital ossifications of snakes. Applying the test of similarity, we conclude that the posterior orbital ossification of snakes is topologically consistent as the homolog of the lacertilian jugal, and that the dorsal orbital ossification present in some snakes (e.g., pythons, Loxocemus, and Calabaria) is the homolog of the lacertilian postfrontal. We therefore propose that the terms postorbital and supraorbital should be abandoned as reference language for the circumorbital bones of snakes, and be replaced with the terms jugal and postfrontal, respectively. The primary homology claim for the snake "postorbital" fails the test of similarity, while the term "supraorbital" is an unnecessary and inaccurate application of the concept of a neomorphic ossification, for an element that passes the test of similarity as a postfrontal. This reinterpretation of the circumorbital bones of snakes is bound to have important repercussions for future phylogenetic analyses and consequently for our understanding of the origin and evolution of snakes. PMID:23630161

  2. [Saccharomyces cerevisiae fungemia in an elderly patient following probiotic treatment].

    PubMed

    Eren, Zehra; Gurol, Yeşim; Sonmezoglu, Meral; Eren, Hatice Seyma; Celik, Gülden; Kantarci, Gülçin

    2014-04-01

    Saccharomyces cerevisiae, known as baker's yeast, is also used as a probiotic agent to treat gastroenteritis by modulating the endogenous flora and immune system. However, since there have been increasing reports of fungemia due to S.cerevisiae and its subspecies S.boulardii, it is recommended that probiotics should be cautiously used in immunosuppressed patients, people with underlying diseases and low-birth weight babies. To emphasize this phenomenon, in this report, a case of S.cerevisiae fungemia developed in a patient given probiotic treatment for antibiotic-associated diarrhea, was presented. An 88-year-old female patient was admitted to our hospital with left hip pain, hypotension, and confusion. Her medical history included hypertension, chronic renal failure, left knee replacement surgery, and recurrent urinary tract infections due to neurogenic bladder. She was transferred to the intensive care unit with the diagnosis of urosepsis. After obtaining blood and urine samples for culture, empirical meropenem (2 x 500 mg) and linezolid (1 x 600 mg) treatment were administered. A central venous catheter (CVC) was inserted and after one day of inotropic support, her hemodynamic parameters were stabilized. The urine culture obtained on admission yielded extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Urine culture was repeated after three days and no bacteria were isolated. On the 4th day of admission she developed diarrhea. Toxin A/B tests for Clostridium difficile were negative. To relieve diarrhea, S.boulardii (Reflor 250 mg capsules, Sanofi Aventis, Turkey) was administered twice a day, without opening capsules. Two days later, her C-reactive protein (CRP) level increased from 23.2 mg/L to 100 mg/L without fever. Her blood culture taken from the CVC yielded S.cerevisiae. Linezolid and meropenem therapies were stopped on the 13th and 14th days, respectively, while prophylactic fluconazole therapy was replaced with

  3. [Saccharomyces cerevisiae fungemia in an elderly patient following probiotic treatment].

    PubMed

    Eren, Zehra; Gurol, Yeşim; Sonmezoglu, Meral; Eren, Hatice Seyma; Celik, Gülden; Kantarci, Gülçin

    2014-04-01

    Saccharomyces cerevisiae, known as baker's yeast, is also used as a probiotic agent to treat gastroenteritis by modulating the endogenous flora and immune system. However, since there have been increasing reports of fungemia due to S.cerevisiae and its subspecies S.boulardii, it is recommended that probiotics should be cautiously used in immunosuppressed patients, people with underlying diseases and low-birth weight babies. To emphasize this phenomenon, in this report, a case of S.cerevisiae fungemia developed in a patient given probiotic treatment for antibiotic-associated diarrhea, was presented. An 88-year-old female patient was admitted to our hospital with left hip pain, hypotension, and confusion. Her medical history included hypertension, chronic renal failure, left knee replacement surgery, and recurrent urinary tract infections due to neurogenic bladder. She was transferred to the intensive care unit with the diagnosis of urosepsis. After obtaining blood and urine samples for culture, empirical meropenem (2 x 500 mg) and linezolid (1 x 600 mg) treatment were administered. A central venous catheter (CVC) was inserted and after one day of inotropic support, her hemodynamic parameters were stabilized. The urine culture obtained on admission yielded extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Urine culture was repeated after three days and no bacteria were isolated. On the 4th day of admission she developed diarrhea. Toxin A/B tests for Clostridium difficile were negative. To relieve diarrhea, S.boulardii (Reflor 250 mg capsules, Sanofi Aventis, Turkey) was administered twice a day, without opening capsules. Two days later, her C-reactive protein (CRP) level increased from 23.2 mg/L to 100 mg/L without fever. Her blood culture taken from the CVC yielded S.cerevisiae. Linezolid and meropenem therapies were stopped on the 13th and 14th days, respectively, while prophylactic fluconazole therapy was replaced with

  4. Rare Homologous Gene Targeting in Histoplasma capsulatum: Disruption of the URA5Hc Gene by Allelic Replacement

    PubMed Central

    Woods, Jon P.; Retallack, Diane M.; Heinecke, Elizabeth L.; Goldman, William E.

    1998-01-01

    URA5 genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Δura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans. PMID:9748447

  5. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    PubMed

    Adeboye, Peter Temitope; Olsson, Lisbeth; Bettiga, Maurizio

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

  6. Protein Remote Homology Detection Based on an Ensemble Learning Approach.

    PubMed

    Chen, Junjie; Liu, Bingquan; Huang, Dong

    2016-01-01

    Protein remote homology detection is one of the central problems in bioinformatics. Although some computational methods have been proposed, the problem is still far from being solved. In this paper, an ensemble classifier for protein remote homology detection, called SVM-Ensemble, was proposed with a weighted voting strategy. SVM-Ensemble combined three basic classifiers based on different feature spaces, including Kmer, ACC, and SC-PseAAC. These features consider the characteristics of proteins from various perspectives, incorporating both the sequence composition and the sequence-order information along the protein sequences. Experimental results on a widely used benchmark dataset showed that the proposed SVM-Ensemble can obviously improve the predictive performance for the protein remote homology detection. Moreover, it achieved the best performance and outperformed other state-of-the-art methods.

  7. Bacterial actin and tubulin homologs in cell growth and division.

    PubMed

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review.

  8. Quantization of gauge fields, graph polynomials and graph homology

    SciTech Connect

    Kreimer, Dirk; Sars, Matthias; Suijlekom, Walter D. van

    2013-09-15

    We review quantization of gauge fields using algebraic properties of 3-regular graphs. We derive the Feynman integrand at n loops for a non-abelian gauge theory quantized in a covariant gauge from scalar integrands for connected 3-regular graphs, obtained from the two Symanzik polynomials. The transition to the full gauge theory amplitude is obtained by the use of a third, new, graph polynomial, the corolla polynomial. This implies effectively a covariant quantization without ghosts, where all the relevant signs of the ghost sector are incorporated in a double complex furnished by the corolla polynomial–we call it cycle homology–and by graph homology. -- Highlights: •We derive gauge theory Feynman from scalar field theory with 3-valent vertices. •We clarify the role of graph homology and cycle homology. •We use parametric renormalization and the new corolla polynomial.

  9. De Novo Sequencing and Homology Searching‡‡*

    PubMed Central

    Ma, Bin; Johnson, Richard

    2012-01-01

    In proteomics, de novo sequencing is the process of deriving peptide sequences from tandem mass spectra without the assistance of a sequence database. Such analyses have traditionally been performed manually by human experts, and more recently by computer programs that have been developed because of the need for higher throughput. Although powerful, de novo sequencing often can only determine partially correct sequence tags because of imperfect tandem mass spectra. However, these sequence tags can then be searched in a sequence database to identify the exact or a homologous peptide. Homology searches are particularly useful for the study of organisms whose genomes have not been sequenced. This tutorial will present background important to understanding de novo sequencing, suggestions on how to do this manually, plus descriptions of computer algorithms used to automate this process and to subsequently carryout homology-based database searches. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 1). PMID:22090170

  10. Protein Remote Homology Detection Based on an Ensemble Learning Approach

    PubMed Central

    Chen, Junjie; Liu, Bingquan; Huang, Dong

    2016-01-01

    Protein remote homology detection is one of the central problems in bioinformatics. Although some computational methods have been proposed, the problem is still far from being solved. In this paper, an ensemble classifier for protein remote homology detection, called SVM-Ensemble, was proposed with a weighted voting strategy. SVM-Ensemble combined three basic classifiers based on different feature spaces, including Kmer, ACC, and SC-PseAAC. These features consider the characteristics of proteins from various perspectives, incorporating both the sequence composition and the sequence-order information along the protein sequences. Experimental results on a widely used benchmark dataset showed that the proposed SVM-Ensemble can obviously improve the predictive performance for the protein remote homology detection. Moreover, it achieved the best performance and outperformed other state-of-the-art methods. PMID:27294123

  11. Remote homology and the functions of metagenomic dark matter.

    PubMed

    Lobb, Briallen; Kurtz, Daniel A; Moreno-Hagelsieb, Gabriel; Doxey, Andrew C

    2015-01-01

    Predicted open reading frames (ORFs) that lack detectable homology to known proteins are termed ORFans. Despite their prevalence in metagenomes, the extent to which ORFans encode real proteins, the degree to which they can be annotated, and their functional contributions, remain unclear. To gain insights into these questions, we applied sensitive remote-homology detection methods to functionally analyze ORFans from soil, marine, and human gut metagenome collections. ORFans were identified, clustered into sequence families, and annotated through profile-profile comparison to proteins of known structure. We found that a considerable number of metagenomic ORFans (73,896 of 484,121, 15.3%) exhibit significant remote homology to structurally characterized proteins, providing a means for ORFan functional profiling. The extent of detected remote homology far exceeds that obtained for artificial protein families (1.4%). As expected for real genes, the predicted functions of ORFans are significantly similar to the functions of their gene neighbors (p < 0.001). Compared to the functional profiles predicted through standard homology searches, ORFans show biologically intriguing differences. Many ORFan-enriched functions are virus-related and tend to reflect biological processes associated with extreme sequence diversity. Each environment also possesses a large number of unique ORFan families and functions, including some known to play important community roles such as gut microbial polysaccharide digestion. Lastly, ORFans are a valuable resource for finding novel enzymes of interest, as we demonstrate through the identification of hundreds of novel ORFan metalloproteases that all possess a signature catalytic motif despite a general lack of similarity to known proteins. Our ORFan functional predictions are a valuable resource for discovering novel protein families and exploring the boundaries of protein sequence space. All remote homology predictions are available at http

  12. Tumor malignancy is engaged to prokaryotic homolog toolbox.

    PubMed

    Fernandes, Janaina; Guedes, Patrícia G; Lage, Celso Luiz S; Rodrigues, Juliany Cola F; Lage, Claudia de Alencar S

    2012-04-01

    Cancer cells display high proliferation rates and survival provided by high glycolysis, chemoresistance and radioresistance, metabolic features that appear to be activated with malignancy, and seemed to have arisen as early in evolution as in unicellular/prokaryotic organisms. Based on these assumptions, we hypothesize that aggressive phenotypes found in malignant cells may be related to acquired unicellular behavior, launched within a tumor when viral and prokaryotic homologs are overexpressed performing likely robust functions. The ensemble of these expressed viral and prokaryotic close homologs in the proteome of a tumor tissue gives them advantage over normal cells. To assess the hypothesis validity, sequences of human proteins involved in apoptosis, energetic metabolism, cell mobility and adhesion, chemo- and radio-resistance were aligned to homologs present in other life forms, excluding all eukaryotes, using PSI-BLAST, with further corroboration from data available in the literature. The analysis revealed that selected sequences of proteins involved in apoptosis and tumor suppression (as p53 and pRB) scored non-significant (E-value>0.001) with prokaryotic homologs; on the other hand, human proteins involved in cellular chemo- and radio-resistance scored highly significant with prokaryotic and viral homologs (as catalase, E-value=zero). We inferred that such upregulated and/or functionally activated proteins in aggressive malignant cells represent a toolbox of modern human homologs evolved from a similar key set that have granted survival of ancient prokaryotes against extremely harsh environments. According to what has been discussed along this analysis, high mutation rates usually hit hotspots in important conserved protein domains, allowing uncontrolled expansion of more resistant, death-evading malignant clones. That is the case of point mutations in key viral proteins affording viruses escape to chemotherapy, and human homologs of such retroviral

  13. Homologous beta-adrenergic desensitization in isolated rat hepatocytes.

    PubMed Central

    García-Sáinz, J A; Michel, B

    1987-01-01

    Hepatocytes from hypothyroid rats have a marked beta-adrenergic responsiveness. Preincubation of these hepatocytes with isoprenaline induced a time-dependent and concentration-dependent desensitization of the beta-adrenergic responsiveness without altering that to glucagon (homologous desensitization). The desensitization was evidenced both in the cyclic AMP accumulation and in the stimulation of ureagenesis induced by the beta-adrenergic agonists. Under the same conditions, preincubation with glucagon induced no desensitization. Propranolol was also unable to induce desensitization, but blocked that induced by isoprenaline. Pertussis-toxin treatment did not alter the homologous beta-adrenergic desensitization induced by isoprenaline. PMID:2825633

  14. [Knock out of bovine beta casein gene by homologous recombination].

    PubMed

    Xue, Ke; Li, Feng; Luo, Guang-Bin; Huang, Wei-Wei; Chen, Xue-Jin

    2007-05-01

    It has been reported that homologous recombination with Red system has been successfully used for knock-out. We try to work on the construction of the expression vector of Mammary Gland with Red system. This study takes CSN2 as a vector for gene target, which contains the complete bovine beta casein gene. Different homologous arms were designed and the CDS region of the beta casein gene was successfully knocked out. The efficiency was also explored for knocking out different DNA fragment. Based on the study, it is very convenient for making a deep research of the foreign gene expression under the regulation of CSN2 flanking region.

  15. Targeted mutagenesis by homologous recombination in D. melanogaster

    PubMed Central

    Rong, Yikang S.; Titen, Simon W.; Xie, Heng B.; Golic, Mary M.; Bastiani, Michael; Bandyopadhyay, Pradip; Olivera, Baldomero M.; Brodsky, Michael; Rubin, Gerald M.; Golic, Kent G.

    2002-01-01

    We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced. PMID:12080094

  16. Ecdysone receptor homologs from mollusks, leeches and a polychaete worm.

    PubMed

    Laguerre, Michel; Veenstra, Jan A

    2010-11-01

    The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes.

  17. The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

    PubMed Central

    Verhage, R; Zeeman, A M; de Groot, N; Gleig, F; Bang, D D; van de Putte, P; Brouwer, J

    1994-01-01

    The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs. Images PMID:8065346

  18. Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains*

    PubMed Central

    Smith, Kathryn D.; Gordon, Patricia B.; Rivetta, Alberto; Allen, Kenneth E.; Berbasova, Tetyana; Slayman, Clifford; Strobel, Scott A.

    2015-01-01

    Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm. PMID:26055717

  19. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs

    SciTech Connect

    Shanklin, J.; Somerville, C. )

    1991-03-15

    Stearoyl-acyl-carrier-protein (ACP) desaturase was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the {Delta}{sup 9} desaturase is developmentally regulated.

  20. Interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae in wine fermentation: influence of inoculation and nitrogen content.

    PubMed

    Taillandier, Patricia; Lai, Quoc Phong; Julien-Ortiz, Anne; Brandam, Cédric

    2014-07-01

    Alcoholic fermentation by an oenological strain of Torulaspora delbrueckii in association with an oenological strain of Saccharomyces cerevisiae was studied in mixed and sequential cultures. Experiments were performed in a synthetic grape must medium in a membrane bioreactor, a special tool designed to study indirect interactions between microorganisms. Results showed that the S. cerevisiae strain had a negative impact on the T. delbrueckii strain, leading to a viability decrease as soon as S. cerevisiae was inoculated. Even for high inoculation of T. delbrueckii (more than 20× S. cerevisiae) in mixed cultures, T. delbrueckii growth was inhibited. Substrate competition and cell-to-cell contact mechanism could be eliminated as explanations of the observed interaction, which was probably an inhibition by a metabolite produced by S. cerevisiae. S. cerevisiae should be inoculated 48 h after T. delbrueckii in order to ensure the growth of T. delbrueckii and consequently a decrease of volatile acidity and a higher isoamyl acetate production. In this case, in a medium with a high concentration of assimilable nitrogen (324 mg L(-1)), S. cerevisiae growth was not affected by T. delbrueckii. But in a sequential fermentation in a medium containing 176 mg L(-1) initial assimilable nitrogen, S. cerevisiae was not able to develop because of nitrogen exhaustion by T. delbrueckii growth during the first 48 h, leading to sluggish fermentation. PMID:24500666

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

    EPA Science Inventory

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T

    PubMed Central

    Kutumbaka, Kirthi K.; Pasmowitz, Joshua; Mategko, James; Reyes, Dindo; Friedrich, Alex; Han, Sukkyun; Martens-Habbena, Willm; Neal-McKinney, Jason; Janagama, Harish K.; Nadala, Cesar

    2015-01-01

    The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1T. PMID:26358606

  4. Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T.

    PubMed

    Kutumbaka, Kirthi K; Pasmowitz, Joshua; Mategko, James; Reyes, Dindo; Friedrich, Alex; Han, Sukkyun; Martens-Habbena, Willm; Neal-McKinney, Jason; Janagama, Harish K; Nadala, Cesar; Samadpour, Mansour

    2015-01-01

    The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1(T). PMID:26358606

  5. [Invertase Overproduction May Provide for Inulin Fermentation by Selection Strains of Saccharomyces cerevisiae].

    PubMed

    Naumov, G I; Naumova, E S

    2015-01-01

    In some recent publications, the ability of selection strains of Saccharomyces cerevisiae to ferment inulin was attributed to inulinase activity. The review summarizes the literature data indicating that overproduction of invertase, an enzyme common to S. cerevisiae, may be responsible for this phenomenon. PMID:26263621

  6. Growth and fermentation characteristics of Saccharomyces cerevisiae NK28 isolated from kiwi fruit.

    PubMed

    Lee, Jong-Sub; Park, Eun-Hee; Kim, Jung-Wan; Yeo, Soo-Hwan; Kim, Myoung-Dong

    2013-09-28

    The influences of glucose concentration, initial medium acidity (pH), and temperature on the growth and ethanol production of Saccharomyces cerevisiae NK28, which was isolated from kiwi fruit, were examined in shake flask cultures. The optimal glucose concentration, initial medium pH, and temperature for ethanol production were 200 g/l, pH 6.0, and 35oC, respectively. Under this growth condition, S. cerevisiae NK28 produced 98.9 ± 5.67 g/l ethanol in 24 h with a volumetric ethanol production rate of 4.12 ± 0.24 g/l·h. S. cerevisiae NK28 was more tolerant to heat and ethanol than laboratory strain S. cerevisiae BY4742, and its tolerance to ethanol and fermentation inhibitors was comparable to that of an ethanologen, S. cerevisiae D5A.

  7. Construction of Killer Industrial Yeast Saccharomyces Cerevisiae Hau-1 and its Fermentation Performance

    PubMed Central

    Bajaj, Bijender K.; Sharma, S.

    2010-01-01

    Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccharomyces cerevisiae MTCC 475, a killer strain. The resultant fusants were characterized for desirable fermentation characteristics. All the technologically important characteristics of distillery yeast Saccharomyces cerevisiae HAU-1 were retained in the fusants, and in addition the killer trait was also introduced into them. Further, the killer activity was found to be stably maintained during hostile conditions of ethanol fermentations in dextrose or molasses, and even during biomass recycling. PMID:24031519

  8. Role of social wasps in Saccharomyces cerevisiae ecology and evolution

    PubMed Central

    Stefanini, Irene; Dapporto, Leonardo; Legras, Jean-Luc; Calabretta, Antonio; Di Paola, Monica; De Filippo, Carlotta; Viola, Roberto; Capretti, Paolo; Polsinelli, Mario; Turillazzi, Stefano; Cavalieri, Duccio

    2012-01-01

    Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain’s evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals. PMID:22847440

  9. An improved method of xylose utilization by recombinant Saccharomyces cerevisiae.

    PubMed

    Ma, Tien-Yang; Lin, Ting-Hsiang; Hsu, Teng-Chieh; Huang, Chiung-Fang; Guo, Gia-Luen; Hwang, Wen-Song

    2012-10-01

    The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-09-01

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

  12. Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

    PubMed Central

    Thomas, D; Surdin-Kerjan, Y

    1997-01-01

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

  13. Differential repair of UV damage in Saccharomyces cerevisiae.

    PubMed

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

    1989-06-26

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

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

    PubMed

    Si, Tong; Zhao, Huimin

    2016-01-01

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

  15. Mating-type gene switching in Saccharomyces cerevisiae.

    PubMed

    Haber, J E

    1998-01-01

    Saccharomyces cerevisiae can change its mating type as often as every generation by a highly choreographed, site-specific recombination event that replaces one MAT allele with different DNA sequences encoding the opposite allele. The study of this process has yielded important insights into the control of cell lineage, the silencing of gene expression, and the formation of heterochromatin, as well as the molecular events of double-strand break-induced recombination. In addition, MAT switching provides a remarkable example of a small locus control region--the Recombination Enhancer--that controls recombination along an entire chromosome arm.

  16. Single molecule detection of direct, homologous, DNA/DNA pairing

    PubMed Central

    Danilowicz, C.; Lee, C. H.; Kim, K.; Hatch, K.; Coljee, V. W.; Kleckner, N.; Prentiss, M.

    2009-01-01

    Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the “default” option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here. PMID:19903884

  17. Multiresolution persistent homology for excessively large biomolecular datasets

    NASA Astrophysics Data System (ADS)

    Xia, Kelin; Zhao, Zhixiong; Wei, Guo-Wei

    2015-10-01

    Although persistent homology has emerged as a promising tool for the topological simplification of complex data, it is computationally intractable for large datasets. We introduce multiresolution persistent homology to handle excessively large datasets. We match the resolution with the scale of interest so as to represent large scale datasets with appropriate resolution. We utilize flexibility-rigidity index to access the topological connectivity of the data set and define a rigidity density for the filtration analysis. By appropriately tuning the resolution of the rigidity density, we are able to focus the topological lens on the scale of interest. The proposed multiresolution topological analysis is validated by a hexagonal fractal image which has three distinct scales. We further demonstrate the proposed method for extracting topological fingerprints from DNA molecules. In particular, the topological persistence of a virus capsid with 273 780 atoms is successfully analyzed which would otherwise be inaccessible to the normal point cloud method and unreliable by using coarse-grained multiscale persistent homology. The proposed method has also been successfully applied to the protein domain classification, which is the first time that persistent homology is used for practical protein domain analysis, to our knowledge. The proposed multiresolution topological method has potential applications in arbitrary data sets, such as social networks, biological networks, and graphs.

  18. Homology and the optimization of DNA sequence data

    NASA Technical Reports Server (NTRS)

    Wheeler, W.

    2001-01-01

    Three methods of nucleotide character analysis are discussed. Their implications for molecular sequence homology and phylogenetic analysis are compared. The criterion of inter-data set congruence, both character based and topological, are applied to two data sets to elucidate and potentially discriminate among these parsimony-based ideas. c2001 The Willi Hennig Society.

  19. Homolog-specific PCR primer design for profiling splice variants

    PubMed Central

    Srivastava, Gyan Prakash; Hanumappa, Mamatha; Kushwaha, Garima; Nguyen, Henry T.; Xu, Dong

    2011-01-01

    To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org. PMID:21415011

  20. A Prominence/filament eruption triggered by eight homologous flares

    NASA Astrophysics Data System (ADS)

    Panesar, Navdeep K.; Sterling, Alphonse; Innes, Davina; Moore, Ronald

    2015-04-01

    Eight homologous flares occurred in active region NOAA 11237 over 16 - 17 June 2011. A prominence system with a surrounding coronal cavity was adjacent to, but still magnetically connected to the active region. The eight eruptions expelled hot material from the active region into the prominence/filament cavity system (PFCS) where the ejecta became confined. We mainly aim to diagnose the 3D dynamics of the PFCS during the series of eight homologous eruptions by using data from two instruments: SDO/AIA and STEREO/EUVI-B, covering the Sun from two directions. The field containing the ejected hot material interacts with the PFCS and causes it to inflate, resulting in a discontinuous rise of the prominence/filament approximately in steps with the homologous eruptions. The eighth eruption triggers the PFCS to move outward slowly, accompanied by a weak coronal dimming. Subsequently the prominence/filament material drains to the solar surface. This PFCS eruption evidently slowly opens field overlying the active region, which results in a final ‘ejective’ eruption from the core of the active region. A strong dimming appears adjacent to the final eruption’s flare loops in the EUVI-B images, followed by a CME. We propose that the eight homologous flares gradually disrupted the PFCS and removed the overlying field above the active region, leading to the CME via the ‘lid removal’ mechanism.

  1. Molecular mechanisms of homologous chromosome pairing and segregation in plants.

    PubMed

    Zhang, Jing; Zhang, Bing; Su, Handong; Birchler, James A; Han, Fangpu

    2014-03-20

    In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division (MI) with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division (MII) with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation. PMID:24656232

  2. On the Homology of Congruence Subgroups and K3(Z)

    PubMed Central

    Lee, Ronnie; Szczarba, R. H.

    1975-01-01

    Let Γ(n;p) be the congruence subgroup of SL(n;Z) of level p. We study the homology and cohomology of Γ(n;p) as modules over SL(n;Fp) and apply our results to obtain an upper bound for the order of K3(Z). PMID:16592224

  3. A Homologation Approach to the Synthesis of Difluorinated Cycloalkynes

    PubMed Central

    2015-01-01

    Difluorinated cyclooctynes are important reagents for labeling azido-biomolecules through copper-free click chemistry. Here, a safe, scalable synthesis of a difluorinated cyclooctyne is reported, which involves a key homologation/ring-expansion reaction. Sequential ring expansions were also employed to synthesize and study a novel difluorinated cyclononyne. PMID:24588780

  4. Multiresolution persistent homology for excessively large biomolecular datasets

    PubMed Central

    Xia, Kelin; Zhao, Zhixiong; Wei, Guo-Wei

    2015-01-01

    Although persistent homology has emerged as a promising tool for the topological simplification of complex data, it is computationally intractable for large datasets. We introduce multiresolution persistent homology to handle excessively large datasets. We match the resolution with the scale of interest so as to represent large scale datasets with appropriate resolution. We utilize flexibility-rigidity index to access the topological connectivity of the data set and define a rigidity density for the filtration analysis. By appropriately tuning the resolution of the rigidity density, we are able to focus the topological lens on the scale of interest. The proposed multiresolution topological analysis is validated by a hexagonal fractal image which has three distinct scales. We further demonstrate the proposed method for extracting topological fingerprints from DNA molecules. In particular, the topological persistence of a virus capsid with 273 780 atoms is successfully analyzed which would otherwise be inaccessible to the normal point cloud method and unreliable by using coarse-grained multiscale persistent homology. The proposed method has also been successfully applied to the protein domain classification, which is the first time that persistent homology is used for practical protein domain analysis, to our knowledge. The proposed multiresolution topological method has potential applications in arbitrary data sets, such as social networks, biological networks, and graphs. PMID:26450288

  5. Disruption of an ADE6 Homolog of Ustilago maydis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ustilago maydis secretes iron-binding compounds during times of iron depletion. A putative homolog of the Sacharromyces cereviseae ADE6 and Escherichia coli purL genes was identified near a multigenic complex, which contains two genes sid1 and sid2 involved in a siderophore biosynthetic pathway. The...

  6. Multiresolution persistent homology for excessively large biomolecular datasets

    SciTech Connect

    Xia, Kelin; Zhao, Zhixiong; Wei, Guo-Wei

    2015-10-07

    Although persistent homology has emerged as a promising tool for the topological simplification of complex data, it is computationally intractable for large datasets. We introduce multiresolution persistent homology to handle excessively large datasets. We match the resolution with the scale of interest so as to represent large scale datasets with appropriate resolution. We utilize flexibility-rigidity index to access the topological connectivity of the data set and define a rigidity density for the filtration analysis. By appropriately tuning the resolution of the rigidity density, we are able to focus the topological lens on the scale of interest. The proposed multiresolution topological analysis is validated by a hexagonal fractal image which has three distinct scales. We further demonstrate the proposed method for extracting topological fingerprints from DNA molecules. In particular, the topological persistence of a virus capsid with 273 780 atoms is successfully analyzed which would otherwise be inaccessible to the normal point cloud method and unreliable by using coarse-grained multiscale persistent homology. The proposed method has also been successfully applied to the protein domain classification, which is the first time that persistent homology is used for practical protein domain analysis, to our knowledge. The proposed multiresolution topological method has potential applications in arbitrary data sets, such as social networks, biological networks, and graphs.

  7. Schizosaccharomyces pombe Sst4p, a Conserved Vps27/Hrs Homolog, Functions Downstream of Phosphatidylinositol 3-Kinase Pik3p To Mediate Proper Spore Formation▿ †

    PubMed Central

    Onishi, Masayuki; Iida, Michihiro; Koga, Takako; Yamada, Sadayuki; Hirata, Aiko; Iwaki, Tomoko; Takegawa, Kaoru; Fukui, Yasuhisa; Tachikawa, Hiroyuki

    2007-01-01

    Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4Δ asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation. PMID:17951524

  8. Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic.

    PubMed

    Brands, Alex; Ho, Tuan-hua David

    2002-11-01

    Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.

  9. BCS1, a novel gene required for the expression of functional Rieske iron-sulfur protein in Saccharomyces cerevisiae.

    PubMed Central

    Nobrega, F G; Nobrega, M P; Tzagoloff, A

    1992-01-01

    Respiratory deficient pet mutants of Saccharomyces cerevisiae assigned to complementation group G2 define a new gene, named BCS1, whose product is shown to be necessary for the expression of functional ubiquinol-cytochrome c reductase (bc1) complex. Immunological assays indicate a gross reduction in the Rieske iron-sulfur subunit in bcs1 mutants, while other subunits of the ubiquinol-cytochrome c reductase complex are present at concentrations comparable to the wild type. Transformation of bcs1 mutants with the iron-sulfur protein gene on a multicopy plasmid led to elevated mitochondrial concentrations of Rieske protein, but did not correct the enzymatic defect, indicating that BCS1 is involved either in forming the active site iron-sulfur cluster or providing a chaperone-like function in assembling the Rieske protein with the other subunits of the complex. Both postulated functions are consistent with the localization of BCS1 in mitochondria. To facilitate further studies on this novel protein, BCS1 was cloned by transformation of a bcs1 mutant and its structure determined. The primary structure of the encoded BCS1 protein bears similarity to a group of proteins that have been implicated in intracellular protein sorting, membrane fusion and regulation of transcription. The region of BCS1 homologous to this diverse group of proteins is approximately 200 amino acids long and includes several signature sequences commonly found in ATPases and nucleotide binding proteins. Images PMID:1327750

  10. Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43.

    PubMed

    Gee, S; Krauss, S W; Miller, E; Aoyagi, K; Arenas, J; Conboy, J G

    1997-10-28

    Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

  11. Genetic alterations leading to increases in internal potassium concentrations are detrimental for DNA integrity in Saccharomyces cerevisiae.

    PubMed

    Merchan, Stephanie; Pedelini, Leda; Hueso, Guillem; Calzada, Arturo; Serrano, Ramón; Yenush, Lynne

    2011-02-01

    We have investigated the effects of alterations in potassium homeostasis on cell cycle progression and genome stability in Saccharomyces cerevisiae. Yeast strains lacking the PPZ1 and PPZ2 phosphatase genes, which aberrantly accumulate potassium, are sensitive to agents causing replicative stress or DNA damage and present a cell cycle delay in the G(1) /S phase. A synthetic slow growth phenotype was identified in a subset of DNA repair mutants upon inhibition of Ppz activity. Moreover, we observe that this slow growth phenotype observed in cdc7(ts) mutants with reduced Ppz activity is reverted by disrupting the TRK1 potassium transporter gene. As over-expression of a mammalian potassium transporter leads to similar phenotypes, we conclude that these defects can be attributed to potassium accumulation. As we reported previously, internal potassium accumulation activates the Slt2 MAP kinase pathway. We show that the removal of SLT2 in ppz1 ppz2 mutants ameliorates sensitivity to agents causing replication stress and DNA damage, whereas over-activation of the pathway leads to similar cell cycle-related defects. Taken together, these results are consistent with inappropriate potassium accumulation reducing DNA replication efficiency, negatively influencing DNA integrity and leading to the requirement of mismatch repair, the MRX complex, or homologous recombination pathways for normal growth.

  12. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    PubMed

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  13. The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

    PubMed Central

    Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

    2013-01-01

    The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3′-end and 5′-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata. PMID:23615469

  14. Crystal Structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

    PubMed Central

    Soniat, Michael; Sampathkumar, Parthasarathy; Collett, Garen; Gizzi, Anthony S.; Banu, Radhika N.; Bhosle, Rahul C.; Chamala, Swetha; Chowdhury, Sukanya; Fiser, Andras; Glenn, Alan S.; Hammonds, James; Hillerich, Brandan; Khafizov, Kamil; Love, James D.; Matikainen, Bridget; Seidel, Ronald D.; Toro, Rafael; Kumar, P. Rajesh; Bonanno, Jeffery B.; Chook, Yuh Min; Almo, Steven C.

    2013-01-01

    Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2•PY-NLS structures. PMID:23535894

  15. Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2.

    PubMed

    Soniat, Michael; Sampathkumar, Parthasarathy; Collett, Garen; Gizzi, Anthony S; Banu, Radhika N; Bhosle, Rahul C; Chamala, Swetha; Chowdhury, Sukanya; Fiser, Andras; Glenn, Alan S; Hammonds, James; Hillerich, Brandan; Khafizov, Kamil; Love, James D; Matikainen, Bridget; Seidel, Ronald D; Toro, Rafael; Rajesh Kumar, P; Bonanno, Jeffery B; Chook, Yuh Min; Almo, Steven C

    2013-06-01

    Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures. PMID:23535894

  16. The expression of Millettia pinnata chalcone isomerase in Saccharomyces cerevisiae salt-sensitive mutants enhances salt-tolerance.

    PubMed

    Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

    2013-01-01

    The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%-86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

  17. Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

    PubMed

    Yin, Yi; Petes, Thomas D

    2013-10-01

    In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

  18. Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae.

    PubMed

    Chiabudini, Marco; Tais, Arlette; Zhang, Ying; Hayashi, Sachiko; Wölfle, Tina; Fitzke, Edith; Rospert, Sabine

    2014-11-01

    Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

  19. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

    PubMed Central

    Drew, David; Newstead, Simon; Sonoda, Yo; Kim, Hyun; von Heijne, Gunnar; Iwata, So

    2009-01-01

    It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment. PMID:18451787

  20. The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae.

    PubMed

    Jandrositz, Anita; Petschnigg, Julia; Zimmermann, Robert; Natter, Klaus; Scholze, Hubert; Hermetter, Albin; Kohlwein, Sepp D; Leber, Regina

    2005-06-15

    Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

  1. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

    SciTech Connect

    Sampathkumar, Parthasarathy; Gheyi, Tarun; Miller, Stacy A.; Bain, Kevin T.; Dickey, Mark; Bonanno, Jeffrey B.; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Burley, Stephen K.

    2012-10-23

    Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

  2. Cloning, primary structure, and regulation of the HIS7 gene encoding a bifunctional glutamine amidotransferase: cyclase from Saccharomyces cerevisiae.

    PubMed

    Kuenzler, M; Balmelli, T; Egli, C M; Paravicini, G; Braus, G H

    1993-09-01

    The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.

  3. A positive but complex association between meiotic double-strand break hotspots and open chromatin in Saccharomyces cerevisiae

    PubMed Central

    Berchowitz, Luke E.; Hanlon, Sean E.; Lieb, Jason D.; Copenhaver, Gregory P.

    2009-01-01

    During meiosis, chromatin undergoes extensive changes to facilitate recombination, homolog pairing, and chromosome segregation. To investigate the relationship between chromatin organization and meiotic processes, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to map open chromatin during the transition from mitosis to meiosis in the budding yeast Saccharomyces cerevisiae. We found that meiosis-induced opening of chromatin is associated with meiotic DSB hotpots. The positive association between open chromatin and DSB hotspots is most prominent 3 h into meiosis, when the early meiotic genes DMC1 and HOP1 exhibit maximum transcription and the early recombination genes SPO11 and RAD51 are strongly up-regulated. While the degree of chromatin openness is positively associated with the occurrence of recombination hotspots, many hotspots occur outside of open chromatin. Of particular interest, many DSB hotspots that fell outside of meiotic open chromatin nonetheless occurred in chromatin that had recently been open during mitotic growth. Finally, we find evidence for meiosis-specific opening of chromatin at the regions adjacent to boundaries of subtelomeric sequences, which exhibit specific crossover control patterns hypothesized to be regulated by chromatin. PMID:19801530

  4. The expression of Millettia pinnata chalcone isomerase in Saccharomyces cerevisiae salt-sensitive mutants enhances salt-tolerance.

    PubMed

    Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

    2013-01-01

    The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%-86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata. PMID:23615469

  5. Oxygen-dependent upstream activation sites of Saccharomyces cerevisiae cytochrome c genes are related forms of the same sequence

    SciTech Connect

    Cerdan, M.E.; Zitomer, R.S.

    1988-06-01

    In Sacchariomyces cerevisiae, the two genes, CYC1 and CYC7, that encode the isoforms of cytochrome c are expressed at different levels. Oxygen regulation is indicated by the expression of the CYP1 gene, and the CYP1 protein interacts with both CYC1 upstream activation sequence 1 (UAS1) and CYC7 UAS/sub 0/. In this study, the homology between the CYP1-binding sites of both genes was investigated. The most noticeable difference between the CYC1 and CYC7 UASs is the presence of GC base pairs at the same positions in a repeated sequence in CYC7 compared with CG base pairs in CYC1. Directed mutagenesis changing these GC residues to CG residues in CYC7 led to CYC1-like expression of CYC7 both in a CYP1 wild-type strain and in a strain carrying the semidominant mutation CYP1-16 which reverses the oxygen-dependent expression of the two genes. The authors' results strongly support the hypothesis that the CYP1-binding sites in CYC1 and CYC7 are related forms of the same sequence and that the CYP1-16 protein has altered specificity for the variant forms of the concensus sequences in both genes.

  6. The putative lipid transporter, Arv1, is required for activating pheromone-induced MAP kinase signaling in Saccharomyces cerevisiae.

    PubMed

    Villasmil, Michelle L; Ansbach, Alison; Nickels, Joseph T

    2011-02-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P(2) and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5(Q59L), which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P(2) polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

  7. The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

    PubMed Central

    Villasmil, Michelle L.; Ansbach, Alison; Nickels, Joseph T.

    2011-01-01

    Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking. PMID:21098723

  8. Structures of the autoproteolytic domain from the Saccharomyces cerevisiae nuclear pore complex component, Nup145

    SciTech Connect

    Sampathkumar, Parthasarathy; Ozyurt, Sinem A.; Do, Johnny; Bain, Kevin T.; Dickey, Mark; Rodgers, Logan A.; Gheyi, Tarun; Sali, Andrej; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sauder, J. Michael; Burley, Stephen K.

    2012-04-30

    Nuclear pore complexes (NPCs) are large, octagonally symmetric dynamic macromolecular assemblies responsible for exchange of proteins and RNAs between the nucleus and cytoplasm. NPCs are made up of at least 456 polypeptides from {approx}30 distinct nucleoporins. Several of these components, sharing similar structural motifs, form stable subcomplexes that form a coaxial structure containing two outer rings (the nuclear and cytoplasmic rings), two inner rings, and a membrane ring. The yeast (Saccharomyces cerevisiae) Nup145 and its human counterpart are unique among the nucleoporins, in that they undergo autoproteolysis to generate functionally distinct proteins. The human counterpart of Nup145 is expressed as two alternatively spliced mRNA transcripts. The larger 190 kDa precursor undergoes post-translational autoproteolysis at the Phe863-Ser864 peptide bond yielding the 92 kDa Nup98 and the 96 kDa Nup96. The smaller 98 kDa precursor is also autoproteolysed at an analogous site giving 92 kDa Nup98-N and a 6 kDa C-terminal fragment, which may form a noncovalent complex. The yeast Nup145 precursor [Fig. 1(A)] contains twelve repeats of a 'GLFG' peptide motif (FG repeats) at its N-terminus, an internal autoproteolytic domain (a region of high conservation with the homologous yeast nucleoporins Nup110 and Nup116, neither of which undergo autoproteolysis), followed by the C-terminal domain. Various forms of the FG repeats are present in nearly half of all nucleoporins; they form intrinsically disordered regions implicated in gating mechanisms that control passage of macromolecules through NPCs. Nup145 undergoes autoproteolysis at the Phe605-Ser606 peptide bond to generate two functionally distinct proteins, Nup145N and Nup145C. Subsequently, Nup145C associates with six other proteins to form the heptameric Y-complex, a component of the outer rings of the NPC. Nup145N, on the other hand, can shuttle between the NPC and the nuclear interior. It has been suggested that Nup

  9. Illustrating and homology modeling the proteins of the Zika virus

    PubMed Central

    Ekins, Sean; Liebler, John; Neves, Bruno J.; Lewis, Warren G.; Coffee, Megan; Bienstock, Rachelle; Southan, Christopher; Andrade, Carolina H.

    2016-01-01

    The Zika virus (ZIKV) is a flavivirus of the family Flaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it either in vitro or in vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our model quality criteria for their further use. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening. PMID:27746901

  10. Heterozygous genome assembly via binary classification of homologous sequence

    PubMed Central

    2015-01-01

    Background Genome assemblers to date have predominantly targeted haploid reference reconstruction from homozygous data. When applied to diploid genome assembly, these assemblers perform poorly, owing to the violation of assumptions during both the contigging and scaffolding phases. Effective tools to overcome these problems are in growing demand. Increasing parameter stringency during contigging is an effective solution to obtaining haplotype-specific contigs; however, effective algorithms for scaffolding such contigs are lacking. Methods We present a stand-alone scaffolding algorithm, ScaffoldScaffolder, designed specifically for scaffolding diploid genomes. The algorithm identifies homologous sequences as found in "bubble" structures in scaffold graphs. Machine learning classification is used to then classify sequences in partial bubbles as homologous or non-homologous sequences prior to reconstructing haplotype-specific scaffolds. We define four new metrics for assessing diploid scaffolding accuracy: contig sequencing depth, contig homogeneity, phase group homogeneity, and heterogeneity between phase groups. Results We demonstrate the viability of using bubbles to identify heterozygous homologous contigs, which we term homolotigs. We show that machine learning classification trained on these homolotig pairs can be used effectively for identifying homologous sequences elsewhere in the data with high precision (assuming error-free reads). Conclusion More work is required to comparatively analyze this approach on real data with various parameters and classifiers against other diploid genome assembly methods. However, the initial results of ScaffoldScaffolder supply validity to the idea of employing machine learning in the difficult task of diploid genome assembly. Software is available at http://bioresearch.byu.edu/scaffoldscaffolder. PMID:25952609

  11. Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination.

    PubMed

    Shao, Renfu; Barker, Stephen C

    2011-02-15

    The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.

  12. DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae.

    PubMed

    Meyer, Damon; Fu, Becky Xu Hua; Heyer, Wolf-Dietrich

    2015-12-15

    Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ. PMID:26607450

  13. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    PubMed

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  14. Biodiversity of Saccharomyces cerevisiae populations in Malbec vineyards from the "Zona Alta del Río Mendoza" region in Argentina.

    PubMed

    Mercado, Laura; Sturm, María Elena; Rojo, María Cecilia; Ciklic, Iván; Martínez, Claudio; Combina, Mariana

    2011-12-15

    The "Zona Alta del Río Mendoza" (ZARM) is the major Malbec grape viticulture region of Argentina. The aim of the present study was to explore Saccharomyces cerevisiae biodiversity in ZARM vineyards. Interdelta PCR and RFLP mtDNA molecular markers were applied to differentiate S. cerevisiae strains. The presence of commercial strains on ZARM vineyards was also assessed. Our results reveal a highly diverse, but genetically closely related, S. cerevisiae population (containing more than 190 molecular patterns among 590 S. cerevisiae isolates). According to the S. cerevisiae strain diversity found in vineyards, they were classified as vineyards with high and low polymorphic S. cerevisiae populations. Six vineyards showed a high polymorphic population, with more than 20 different S. cerevisiae molecular patterns. S. cerevisiae populations in these vineyards were diverse and irregularly distributed, with different strains in each vineyard site. Low polymorphic S. cerevisiae population vineyards displayed very low yeast diversity, with only 9 to 10 different S. cerevisiae strains and presence of two commercial strains widely distributed. Population diversity estimators were calculated to determine the population structure of S. cerevisiae in the ZARM vineyards. The obtained values support the hypothesis that the eight sampled subpopulations come indeed from a larger population.

  15. Genomic evolution of Saccharomyces cerevisiae under Chinese rice wine fermentation.

    PubMed

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

    2014-09-10

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

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

    PubMed

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

    2016-09-20

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

  17. Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress.

    PubMed

    Modig, Tobias; Granath, Katarina; Adler, Lennart; Lidén, Gunnar

    2007-05-01

    Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.

  18. Proteomic analysis of Saccharomyces cerevisiae under high gravity fermentation conditions.

    PubMed

    Pham, Trong Khoa; Chong, Poh Kuan; Gan, Chee Sian; Wright, Phillip C

    2006-12-01

    Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.

  19. Quantifying separation and similarity in a Saccharomyces cerevisiae metapopulation

    PubMed Central

    Knight, Sarah; Goddard, Matthew R

    2015-01-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2016-04-01

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

  3. Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae

    PubMed Central

    Possner, Dominik D. D.; Claesson, Magnus; Guy, Jodie E.

    2015-01-01

    In the yeast Saccharomyces cerevisiae, members of the Kre2/Mnt1 protein family have been shown to be α-1,2-mannosyltransferases or α-1,2-mannosylphosphate transferases, utilising an Mn2+-coordinated GDP-mannose as the sugar donor and a variety of mannose derivatives as acceptors. Enzymes in this family are localised to the Golgi apparatus, and have been shown to be involved in both N- and O-linked glycosylation of newly-synthesised proteins, including cell wall glycoproteins. Our knowledge of the nine proteins in this family is however very incomplete at present. Only one family member, Kre2p/Mnt1p, has been studied by structural methods, and three (Ktr4p, Ktr5p, Ktr7p) are completely uncharacterised and remain classified only as putative glycosyltransferases. Here we use in vitro enzyme activity assays to provide experimental confirmation of the predicted glycosyltransferase activity of Ktr4p. Using GDP-mannose as the donor, we observe activity towards the acceptor methyl-α-mannoside, but little or no activity towards mannose or α-1,2-mannobiose. We also present the structure of the lumenal catalytic domain of S. cerevisiae Ktr4p, determined by X-ray crystallography to a resolution of 2.2 Å, and the complex of the enzyme with GDP to 1.9 Å resolution. PMID:26296208

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2009-08-01

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

  6. Computational models reveal genotype-phenotype associations in Saccharomyces cerevisiae.

    PubMed

    Franco-Duarte, Ricardo; Mendes, Inês; Umek, Lan; Drumonde-Neves, João; Zupan, Blaz; Schuller, Dorit

    2014-07-01

    Genome sequencing is essential to understand individual variation and to study the mechanisms that explain relations between genotype and phenotype. The accumulated knowledge from large-scale genome sequencing projects of Saccharomyces cerevisiae isolates is being used to study the mechanisms that explain such relations. Our objective was to undertake genetic characterization of 172 S. cerevisiae strains from different geographical origins and technological groups, using 11 polymorphic microsatellites, and computationally relate these data with the results of 30 phenotypic tests. Genetic characterization revealed 280 alleles, with the microsatellite ScAAT1 contributing most to intrastrain variability, together with alleles 20, 9 and 16 from the microsatellites ScAAT4, ScAAT5 and ScAAT6. These microsatellite allelic profiles are characteristic for both the phenotype and origin of yeast strains. We confirm the strength of these associations by construction and cross-validation of computational models that can predict the technological application and origin of a strain from the microsatellite allelic profile. Associations between microsatellites and specific phenotypes were scored using information gain ratios, and significant findings were confirmed by permutation tests and estimation of false discovery rates. The phenotypes associated with higher number of alleles were the capacity to resist to sulphur dioxide (tested by the capacity to grow in the presence of potassium bisulphite) and the presence of galactosidase activity. Our study demonstrates the utility of computational modelling to estimate a strain technological group and phenotype from microsatellite allelic combinations as tools for preliminary yeast strain selection.

  7. [Rule of homology and morbid anatomy (author's transl)].

    PubMed

    Doerr, W

    1979-07-27

    1. According to J.W. Goethe, morphology is a theory of evolution, H. Braus defined it as a theory of historic incidents, and according to D. Starck morphology is the role of shapes of the organisms. 2. The term homology was coined by morphologic researchers. Of course, it is used nowadays also in mathematics, chemistry, and linguistics and other logic matters. 3. Homologies have a special position in Goethe's work on the theory of types. Goethe's morphologic research and Schiller's aesthetic speculations are considered to be the origin of a 'typologic point of view.' 4. Coherences of Platon's theory of ideas and Goethe's theory of types are scrutinized. The theory of shapes ('Gestalt theory') is inconceivable without Platon's theory, and scientic morphology is inconceivable without shapes, either, and according to C. v. Ehrenfels "Gestaltphilosophie" could not exist without the shapes of Platon's theory. 5. It is shown that without Gestalt philosophy one cannot comprehend the following coherences: Gestalt (shape) as an idea, idea as a type of Goethe's rule, type as an element of the theory of homologies and even of constitution. 6. Homology will be constituted using certain criterions: a) detection of an equal descent, b) equal position of organismic structures in individuals, c) evidence of interpositions, and d) certain qualities of parts which are compared with each other. Homologous structures may be dissimilar in their architecture. 7. The term homology is explained a) by giving an analysis of morphologic and teratologic lines, b) by scrutinizing froms of symmetry, and c) by presenting the histopathology of topographical diverse but according to the morphogenetic mode coinciding tumours which are resembling each other in their microscopic patterns. 8. The application of the rule of homology in the morphologic investigation of diseases proves to be a) valuable from a heuristic point of view, b) an instrument of communication to characterize comparable matters

  8. Fumaric Acid Production in Saccharomyces cerevisiae by In Silico Aided Metabolic Engineering

    PubMed Central

    Xu, Guoqiang; Zou, Wei; Chen, Xiulai; Xu, Nan; Liu, Liming; Chen, Jian

    2012-01-01

    Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L–1 without any apparent change in growth in fed-batch culture. FT-IR and 1H and 13C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L–1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L–1 FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering. PMID:23300594

  9. Inactivation of Saccharomyces cerevisiae suspended in orange juice using high-intensity pulsed electric fields.

    PubMed

    Elez-Martínez, Pedro; Escolà-Hernández, Joan; Soliva-Fortuny, Robert C; Martín-Belloso, Olga

    2004-11-01

    Saccharomyces cerevisiae is often associated with the spoilage of fruit juices. The purpose of this study was to evaluate the effect of high-intensity pulsed electric field (HIPEF) treatment on the survival of S. cerevisiae suspended in orange juice. Commercial heat-sterilized orange juice was inoculated with S. cerevisiae (CECT 1319) (10(8) CFU/ml) and then treated by HIPEFs. The effects of HIPEF parameters (electric field strength, treatment time, pulse polarity, frequency, and pulse width) were evaluated and compared to those of heat pasteurization (90 degrees C/min). In all of the HIPEF experiments, the temperature was kept below 39 degrees C. S. cerevisiae cell damage induced by HIPEF treatment was observed by electron microscopy. HIPEF treatment was effective for the inactivation of S. cerevisiae in orange juice at pasteurization levels. A maximum inactivation of a 5.1-log (CFU per milliliter) reduction was achieved after exposure of S. cerevisiae to HIPEFs for 1,000 micros (4-micros pulse width) at 35 kV/cm and 200 Hz in bipolar mode. Inactivation increased as both the field strength and treatment time increased. For the same electric field strength and treatment time, inactivation decreased when the frequency and pulse width were increased. Electric pulses applied in the bipolar mode were more effective than those in the monopolar mode for destroying S. cerevisiae. HIPEF processing inactivated S. cerevisiae in orange juice, and the extent of inactivation was similar to that obtained during thermal pasteurization. HIPEF treatments caused membrane damage and had a profound effect on the intracellular organization of S. cerevisiae.

  10. Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

    PubMed

    Umeda, F; Nishikawa, T; Miyasaka, H; Maeda, I; Kawase, M; Yagi, K

    2001-11-01

    Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens. PMID:11916262

  11. Molecular evolution of a Drosophila homolog of human BRCA2

    PubMed Central

    Bennett, Sarah M.; Noor, Mohamed A. F.

    2009-01-01

    The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region (“BRC repeats”) with RAD-51. A putatively simpler homolog, dmbrca2, was identified in Drosophila melanogaster recently and also affects mitotic and meiotic double-strand break repair. In this study, we examined patterns of repeat variation both within Drosophila pseudoobscura and among available Drosophila genome sequences. We identified extensive variation within and among closely related Drosophila species in BRC repeat number, to the extent that variation within this genus recapitulates the extent of variation found across the entire animal kingdom. We describe patterns of evolution across species by documenting recent repeat expansions (sometimes in tandem arrays) and homogenizations within available genome sequences. Overall, we have documented patterns and modes of evolution in a new model system of a gene which is important to human health. PMID:19554456

  12. Back-Translation for Discovering Distant Protein Homologies

    NASA Astrophysics Data System (ADS)

    Gîrdea, Marta; Noé, Laurent; Kucherov, Gregory

    Frameshift mutations in protein-coding DNA sequences produce a drastic change in the resulting protein sequence, which prevents classic protein alignment methods from revealing the proteins’ common origin. Moreover, when a large number of substitutions are additionally involved in the divergence, the homology detection becomes difficult even at the DNA level. To cope with this situation, we propose a novel method to infer distant homology relations of two proteins, that accounts for frameshift and point mutations that may have affected the coding sequences. We design a dynamic programming alignment algorithm over memory-efficient graph representations of the complete set of putative DNA sequences of each protein, with the goal of determining the two putative DNA sequences which have the best scoring alignment under a powerful scoring system designed to reflect the most probable evolutionary process. This allows us to uncover evolutionary information that is not captured by traditional alignment methods, which is confirmed by biologically significant examples.

  13. Molecular evolution of a Drosophila homolog of human BRCA2.

    PubMed

    Bennett, Sarah M; Noor, Mohamed A F

    2009-11-01

    The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region ("BRC repeats") with RAD-51. A putatively simpler homolog, dmbrca2, was identified in Drosophila melanogaster recently and also affects mitotic and meiotic double-strand break repair. In this study, we examined patterns of repeat variation both within Drosophila pseudoobscura and among available Drosophila genome sequences. We identified extensive variation within and among closely related Drosophila species in BRC repeat number, to the extent that variation within this genus recapitulates the extent of variation found across the entire animal kingdom. We describe patterns of evolution across species by documenting recent repeat expansions (sometimes in tandem arrays) and homogenizations within available genome sequences. Overall, we have documented patterns and modes of evolution in a new model system of a gene which is important to human health.

  14. Homology and isomorphism: Bourdieu in conversation with New Institutionalism.

    PubMed

    Wang, Yingyao

    2016-06-01

    Bourdieusian Field Theory (BFT) provided decisive inspiration for the early conceptual formulation of New Institutionalism (NI). This paper attempts to reinvigorate the stalled intellectual dialogue between NI and BFT by comparing NI's concept of isomorphism with BFT's notion of homology. I argue that Bourdieu's understanding of domination-oriented social action, transposable habitus, and a non-linear causality, embodied in his neglected concept of homology, provides an alternative theorization of field-level convergence to New Institutionalism's central idea of institutional isomorphism. To showcase how BFT can be useful for organizational research, I postulate a habitus-informed and field-conditioned theory of transference to enrich NI's spin-off thesis of 'diffusion'. I propose that while NI can benefit from BFT's potential of bringing social structure back into organizational research, BFT can enrich its social analysis by borrowing from NI's elaboration of the symbolic system of organizations.

  15. Homology and isomorphism: Bourdieu in conversation with New Institutionalism.

    PubMed

    Wang, Yingyao

    2016-06-01

    Bourdieusian Field Theory (BFT) provided decisive inspiration for the early conceptual formulation of New Institutionalism (NI). This paper attempts to reinvigorate the stalled intellectual dialogue between NI and BFT by comparing NI's concept of isomorphism with BFT's notion of homology. I argue that Bourdieu's understanding of domination-oriented social action, transposable habitus, and a non-linear causality, embodied in his neglected concept of homology, provides an alternative theorization of field-level convergence to New Institutionalism's central idea of institutional isomorphism. To showcase how BFT can be useful for organizational research, I postulate a habitus-informed and field-conditioned theory of transference to enrich NI's spin-off thesis of 'diffusion'. I propose that while NI can benefit from BFT's potential of bringing social structure back into organizational research, BFT can enrich its social analysis by borrowing from NI's elaboration of the symbolic system of organizations. PMID:27218878

  16. Levels of homology and the problem of neocortex.

    PubMed

    Dugas-Ford, Jennifer; Ragsdale, Clifton W

    2015-07-01

    The neocortex is found only in mammals, and the fossil record is silent on how this soft tissue evolved. Understanding neocortex evolution thus devolves to a search for candidate homologous neocortex traits in the extant nonmammalian amniotes. The difficulty is that homology is based on similarity, and the six-layered neocortex structure could hardly be more dissimilar in appearance from the nuclear organization that is so conspicuous in the dorsal telencephalon of birds and other reptiles. Recent molecular data have, however, provided new support for one prominent hypothesis, based on neuronal circuits, that proposes the principal neocortical input and output cell types are a conserved feature of amniote dorsal telencephalon. Many puzzles remain, the greatest being understanding the selective pressures and molecular mechanisms that underlie such tremendous morphological variation in telencephalon structure. PMID:26154980

  17. The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

    PubMed Central

    Sakuraba, Yasuhito; Park, So-Yon; Paek, Nam-Chon

    2015-01-01

    Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs. PMID:25913011

  18. Yeast Saccharomyces cerevisiae adiponectin receptor homolog Izh2 is involved in the regulation of zinc, phospholipid and pH homeostasis.

    PubMed

    Mattiazzi Ušaj, Mojca; Prelec, Metod; Brložnik, Mojca; Primo, Cecilia; Curk, Tomaž; Ščančar, Janez; Yenush, Lynne; Petrovič, Uroš

    2015-09-01

    The functional link between zinc homeostasis and membrane-related processes, including lipid metabolism regulation, extends from yeast to humans, and has a likely role in the pathogenesis of diabetes. The yeast Izh2 protein has been previously implicated in zinc ion homeostasis and in the regulation of lipid and phosphate metabolism, but its precise molecular function is not known. We performed a chemogenomics experiment to determine the genes conferring resistance or sensitivity to different environmental zinc concentrations. We then determined at normal, depleted and excess zinc concentrations, the genetic interactions of IZH2 at the genome-wide level and measured changes in the transcriptome caused by deletion of IZH2. We found evidence for an important cellular function of the Rim101 pathway in zinc homeostasis in neutral or acidic environments, and observed that phosphatidylinositol is a source of inositol when zinc availability is limited. Comparison of our experimental profiles with published gene expression and genetic interaction profiles revealed pleiotropic functions for Izh2. We propose that Izh2 acts as an integrator of intra- and extracellular signals in providing adequate cellular responses to maintain homeostasis under different external conditions, including - but not limited to - alterations in zinc concentrations.

  19. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

  20. Serum Anti-Saccharomyces Cerevisiae Antibodies in Greek Patients with Behcet's Disease

    PubMed Central

    Vaiopoulos, George; Lakatos, Peter Laszlo; Papp, Maria; Kaklamanis, Faedon; Economou, Efrosyni; Zevgolis, Vassilis; Sourdis, John

    2011-01-01

    We tested 59 Greek patients with Behcet's Disease (BD) for serum anti-Saccharomyces cerevisiae antibodies. No increase of these antibodies was detected in the cases compared to 55 healthy unrelated blood donors from the same population. This finding is in contrast with the correlation between Saccharomyces cerevisiae antibodies and BD as reported in other populations. It seems that environmental factors may contribute to disease expression in different populations, producing different effects according to the individual's genetic predisposition. Saccharomyces cerevisiae antibodies do not seem to be of any significance in the Greek population. PMID:21319357