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Sample records for cerevisiae prm1 homolog

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

    PubMed

    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

  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. Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system

    SciTech Connect

    Ahn, B.Y.; Dornfeld, K.J.; Fagrelius, T.J.; Livingston, D.M.

    1988-06-01

    Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conservation in mitotically dividing S. cerevisiae cels. The authors used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10/sup -3/ event soper cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergones gene conversion at a rate of approximately 1 x 10/sup -10/ events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

  4. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  5. Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

    PubMed Central

    Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

    2014-01-01

    The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  6. A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.

    PubMed Central

    Kim, S I; Stange-Thomann, N; Martins, O; Hong, K W; Söll, D; Fox, T D

    1997-01-01

    A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation. PMID:9287027

  7. 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

  8. Novel human and mouse homologs of Saccharomyces cerevisiae DNA polymerase eta.

    PubMed

    McDonald, J P; Rapić-Otrin, V; Epstein, J A; Broughton, B C; Wang, X; Lehmann, A R; Wolgemuth, D J; Woodgate, R

    1999-08-15

    The Saccharomyces cerevisiae RAD30 gene encodes a novel eukaryotic DNA polymerase, pol eta that is able to replicate across cis-syn cyclobutane pyrimidine dimers both accurately and efficiently. Very recently, a human homolog of RAD30 was identified, mutations in which result in the sunlight-sensitive, cancer-prone, Xeroderma pigmentosum variant group phenotype. We report here the cloning and localization of a second human homolog of RAD30. Interestingly, RAD30B is localized on chromosome 18q21.1 in a region that is often implicated in the etiology of many human cancers. The mouse homolog (Rad30b) is located on chromosome 18E2. The human RAD30B and mouse Rad30b mRNA transcripts, like many repair proteins, are highly expressed in the testis. In situ hybridization analysis indicates that expression of mouse Rad30b occurs predominantly in postmeiotic round spermatids. Database searches revealed genomic and EST sequences from other eukaryotes such as Aspergillus nidulans, Schizosaccharomyces pombe, Brugia malayi, Caenorhabditis elegans, Trypanosoma cruzi, Arabidopsis thaliana, and Drosophila melanogaster that also encode putative homologs of RAD30, thereby suggesting that Rad30-dependent translesion DNA synthesis is conserved within the eukaryotic kingdom. PMID:10458907

  9. Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae.

    PubMed

    Martins, L J; Jensen, L T; Simon, J R; Keller, G L; Winge, D R; Simons, J R

    1998-09-11

    The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in the S. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G). PMID:9726978

  10. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination

    PubMed Central

    Bonnefoy, Nathalie; Fox, Thomas D.

    2009-01-01

    Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mtDNA. DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. While transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes, and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology. PMID:18314724

  11. Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

    PubMed Central

    Lee, Min-Soo; Yu, Mi; Kim, Kyoung-Yeon; Park, Geun-Hee; Kwack, KyuBum; Kim, Keun P.

    2015-01-01

    Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer. PMID:25938495

  12. 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

  13. Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae.

    PubMed Central

    Shor, Erika; Gangloff, Serge; Wagner, Marisa; Weinstein, Justin; Price, Gavrielle; Rothstein, Rodney

    2002-01-01

    In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Delta does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage. PMID:12399378

  14. 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

  15. Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae.

    PubMed Central

    Butler, David K; Gillespie, David; Steele, Brandi

    2002-01-01

    Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1. PMID:12136011

  16. La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth.

    PubMed Central

    Yoo, C J; Wolin, S L

    1994-01-01

    The human autoantigen La is a 50-kDa protein which binds to the 3' termini of virtually all nascent polymerase III transcripts. Experiments with mammalian transcription extracts have led to the proposal that the La protein is required for multiple rounds of transcription by RNA polymerase III (E. Gottlieb and J. A. Steitz, EMBO J. 8:851-861, 1989; R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Although La protein homologs have been identified in a variety of vertebrate species, the protein has not been identified in invertebrates. In order to begin a genetic analysis of La protein function, we have characterized homologs of the La protein in the fruit fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae. We show that both the Drosophila and yeast La proteins are bound to precursors of polymerase III RNAs in vivo. The Drosophila and yeast proteins resemble the human La protein in their biochemical properties, as both proteins can be partially purified from cells by a procedure previously devised to purify the human protein. Similarly to vertebrate La proteins, the Drosophila and yeast homologs preferentially bind RNAs that terminate with a 3' hydroxyl. Despite the fact that the La protein is conserved between humans and Saccharomyces cerevisiae, yeast cells containing a null allele of the gene encoding the La protein are viable, suggesting that another protein(s) plays a functionally redundant role. Images PMID:8035818

  17. Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts.

    PubMed Central

    Costanzo, M C; Bonnefoy, N; Williams, E H; Clark-Walker, G D; Fox, T D

    2000-01-01

    Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous. PMID:10757749

  18. 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

  19. The Plasma Membrane Proteins Prm1 and Fig1 Ascertain Fidelity of Membrane Fusion during Yeast Mating

    PubMed Central

    Engel, Alex; Walter, Peter

    2007-01-01

    As for most cell–cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca2+ influx, yields similar cell fusion defects. Although extracellular Ca2+ is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca2+ is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca2+ binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca2+ is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca2+-dependent membrane repair. PMID:17151357

  20. Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Hong, Soogil; Kim, Keun Pil

    2013-01-01

    Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias. PMID:24213600

  1. 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

  2. Dynamics of Homology Searching During Gene Conversion in Saccharomyces cerevisiae Revealed by Donor Competition

    PubMed Central

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

    2011-01-01

    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. PMID:21954161

  3. The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

    PubMed

    Ye, Yanfang; Kirkham-McCarthy, Lucy; Lahue, Robert S

    2016-07-01

    Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11. PMID:27173583

  4. Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae

    PubMed Central

    Rubio-Texeira, Marta; Varnum, James M; Bieganowski, Pawel; Brenner, Charles

    2002-01-01

    Background The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown. Results Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells. Conclusions The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2. PMID:12028594

  5. Yeast ribosomal proteins: XIII. Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7.

    PubMed Central

    Mizuta, K; Hashimoto, T; Otaka, E

    1992-01-01

    We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae. It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI. The haploid strains carrying disrupted YL8A grew more slowly than the parent strain. The open reading frame is interrupted with two introns. The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others. Images PMID:1549461

  6. 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

  7. Association study of six SNPs in PRM1, PRM2 and TNP2 genes in iranian infertile men with idiopathic azoospermia

    PubMed Central

    Siasi, Elham; Aleyasin, Ahmad; Mowla, Javad; Sahebkashaf, Hamid

    2012-01-01

    Background: Histones are replaced by protamines to condensate and package DNA into the sperm head during mammalian spermatogenesis. Protamine genes defects have been reported to cause sperm DNA damage and male infertility. Objective: In this study relationship among some protamines genes family SNPs include PRM1 (C321A), PRM2 (C248T) and TNP2 (T1019C), (G1272C), (G del in 1036 and 1046 bp) were studied in 96 idiopathic infertile men with azoospermia or oligospermia and 100 normal control men. Materials and Methods: Analysis of SNPs was performed using restriction fragment length polymorphism (PCR-RFLP), single strand conformational polymorphism (PCR-SSCP) and PCR sequencing. Results: No polymorphisms were found for tested SNPs except for PRM1 (C321A) and TNP2 (G1272C) in which frequency of altered AA and GG genotypes were slightly higher in infertile case group. Statistical analysis showed no significant association related to PRM1 (C321A) p=0.805 and TNP2 (G1272C) loci p=0.654. Conclusion: These results are consistent with previous studies and indicating that all tested SNPs was not associated with oligospermia and azospermia and idiopatic male infertility in Iranian population. PMID:25246894

  8. 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

  9. KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae.

    PubMed

    Staneva, Dessislava; Uccelletti, Daniela; Farina, Francesca; Venkov, Pencho; Palleschi, Claudio

    2004-01-15

    Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity. PMID:14745781

  10. The Candida boidinii peroxisomal membrane protein Pmp30 has a role in peroxisomal proliferation and is functionally homologous to Pmp27 from Saccharomyces cerevisiae.

    PubMed Central

    Sakai, Y; Marshall, P A; Saiganji, A; Takabe, K; Saiki, H; Kato, N; Goodman, J M

    1995-01-01

    The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization. PMID:7592467

  11. 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

  12. The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination.

    PubMed Central

    Houston, Peter; Simon, Peter J; Broach, James R

    2004-01-01

    Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway. PMID:15082540

  13. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae

    PubMed Central

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-01-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  14. The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

    PubMed

    Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

    2016-07-01

    Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

  15. Quantification of sperm specific mRNA transcripts (PRM1, PRM2, and TNP2) in teratozoospermia and normozoospermia: New correlations between mRNA content and morphology of sperm.

    PubMed

    Savadi-Shiraz, Elham; Edalatkhah, Haleh; Talebi, Saeed; Heidari-Vala, Hamed; Zandemami, Mahdi; Pahlavan, Somayeh; Modarressi, Mohammad Hossein; Akhondi, Mohammad Mehdi; Paradowska-Dogan, Agnieszka; Sadeghi, Mohammad Reza

    2015-01-01

    Sperm mRNAs could be used as a predictor of fertilization capacity since the transcriptional profile of a gamete is critical for the production of viable human sperm. The aim of this study was to determine if PRM1, PRM2, and TNP2 transcripts in spermatozoa from normozoospermic and teratozoospermic men correlate with sperm morphology and/or assisted-reproduction outcomes. Human ejaculates were collected from 138 men referred to an infertility clinic, and were separated in two groups, teratozoospermic (n =72) and normozoospermic (n =66), based on World Health Organization criteria (2010). Chromomycin A3 and analine blue staining were used to evaluate protamination and chromatin integrity, respectively. Quantitative reverse-transcriptase PCR was performed for PRM1, PRM2, and TNP2. This analysis revealed significantly higher PRM1 and PRM2 mRNA copy numbers in normozoospermic versus teratozoospermic samples (P < 0.001). In contrast, TNP2 transcript abundance was significantly higher in teratozoospermic versus normozoospermic samples (P < 0.001) and positively correlated with sperm-head defects (P < 0.05). Sperm-tail defects negatively correlated (P < 0.05) with both PRM1 and PRM2 transcripts in normozoospermic samples. No significant differences were observed between the two groups when comparing transcript levels to the outcome of intracytoplasmic sperm injection cycles (P > 0.05), and a normal PRM1/PRM2 mRNA ratio (∼1) was observed in more than 70% of successful cycles. Thus, the quantity of PRM1, PRM2, and TNP2 transcripts and the PRM1/PRM2 mRNA ratio affect spermiogenesis, sperm morphology, and the function of mature human sperm. These mRNAs could therefore be used as biomarkers for the diagnosis of male infertility. PMID:25536093

  16. BTN1, the Saccharomyces cerevisiae homolog to the human Batten disease gene, is involved in phospholipid distribution

    PubMed Central

    Padilla-López, Sergio; Langager, Deanna; Chan, Chun-Hung; Pearce, David A.

    2012-01-01

    SUMMARY BTN1, the yeast homolog to human CLN3 (which is defective in Batten disease), has been implicated in the regulation of vacuolar pH, potentially by modulating vacuolar-type H+-ATPase (V-ATPase) activity. However, we report that Btn1p and the V-ATPase complex do not physically interact, suggesting that any influence that Btn1p has on V-ATPase is indirect. Because membrane lipid environment plays a crucial role in the activity and function of membrane proteins, we investigated whether cells lacking BTN1 have altered membrane phospholipid content. Deletion of BTN1 (btn1-Δ) led to a decreased level of phosphatidylethanolamine (PtdEtn) in both mitochondrial and vacuolar membranes. In yeast there are two phosphatidylserine (PtdSer) decarboxylases, Psd1p and Psd2p, and these proteins are responsible for the synthesis of PtdEtn in mitochondria and Golgi-endosome, respectively. Deletion of both BTN1 and PSD1 (btn1-Δ psd1-Δ) led to a further decrease in levels of PtdEtn in ER membranes associated to mitochondria (MAMs), with a parallel increase in PtdSer. Fluorescent-labeled PtdSer (NBD-PtdSer) transport assays demonstrated that transport of NBD-PtdSer from the ER to both mitochondria and endosomes and/or vacuole is affected in btn1-Δ cells. Moreover, btn1-Δ affects the synthesis of PtdEtn by the Kennedy pathway and impairs the ability of psd1-Δ cells to restore PtdEtn to normal levels in mitochondria and vacuoles by ethanolamine addition. In summary, lack of Btn1p alters phospholipid levels and might play a role in regulating their subcellular distribution. PMID:22107873

  17. BTN1, the Saccharomyces cerevisiae homolog to the human Batten disease gene, is involved in phospholipid distribution.

    PubMed

    Padilla-López, Sergio; Langager, Deanna; Chan, Chun-Hung; Pearce, David A

    2012-03-01

    BTN1, the yeast homolog to human CLN3 (which is defective in Batten disease), has been implicated in the regulation of vacuolar pH, potentially by modulating vacuolar-type H(+)-ATPase (V-ATPase) activity. However, we report that Btn1p and the V-ATPase complex do not physically interact, suggesting that any influence that Btn1p has on V-ATPase is indirect. Because membrane lipid environment plays a crucial role in the activity and function of membrane proteins, we investigated whether cells lacking BTN1 have altered membrane phospholipid content. Deletion of BTN1 (btn1-Δ) led to a decreased level of phosphatidylethanolamine (PtdEtn) in both mitochondrial and vacuolar membranes. In yeast there are two phosphatidylserine (PtdSer) decarboxylases, Psd1p and Psd2p, and these proteins are responsible for the synthesis of PtdEtn in mitochondria and Golgi-endosome, respectively. Deletion of both BTN1 and PSD1 (btn1-Δ psd1-Δ) led to a further decrease in levels of PtdEtn in ER membranes associated to mitochondria (MAMs), with a parallel increase in PtdSer. Fluorescent-labeled PtdSer (NBD-PtdSer) transport assays demonstrated that transport of NBD-PtdSer from the ER to both mitochondria and endosomes and/or vacuole is affected in btn1-Δ cells. Moreover, btn1-Δ affects the synthesis of PtdEtn by the Kennedy pathway and impairs the ability of psd1-Δ cells to restore PtdEtn to normal levels in mitochondria and vacuoles by ethanolamine addition. In summary, lack of Btn1p alters phospholipid levels and might play a role in regulating their subcellular distribution. PMID:22107873

  18. 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.

  19. A Homolog of Ste6, the a-Factor Transporter in Saccharomyces cerevisiae, Is Required for Mating but Not for Monokaryotic Fruiting in Cryptococcus neoformans

    PubMed Central

    Hsueh, Yen-Ping; Shen, Wei-Chiang

    2005-01-01

    Fungal pheromones function during the initial recognition stage of the mating process. One type of peptide pheromone identified in ascomycetes and basidiomycetes terminates in a conserved CAAX motif and requires extensive posttranslational modifications to become mature and active. A well-studied representative is the a-factor of Saccharomyces cerevisiae. Unlike the typical secretory pathway utilized by most peptides, an alternative mechanism involving the ATP-binding cassette transporter Ste6 is used for the export of mature a-factor. Cryptococcus neoformans, a bipolar human pathogenic basidiomycete, produces CAAX motif-containing lipopeptide pheromones in both MATa and MATα cells. Virulence studies with a congenic pair of C. neoformans serotype D strains have shown that MATα cells are more virulent than MATa cells. Characterization of the MATα pheromones indicated that an autocrine signaling loop may contribute to the differentiation and virulence of MATα cells. To further address the role of pheromones in the signaling loop, we identified a STE6 homolog in the C. neoformans genome and determined its function by gene disruption. The ste6 mutants in either mating-type background showed partially impaired mating functions, and mating was completely abolished in a bilateral mutant cross. Surprisingly, the MATα ste6 mutant does not exhibit a defect in monokaryotic fruiting, suggesting that the activation of the autocrine signaling loop by the pheromone is via a Ste6-independent mechanism. MFα pheromone itself is essential for this process and could induce the signaling response intracellularly in MATα cells. Our data demonstrate that Ste6 is evolutionarily conserved for mating and is not required for monokaryotic fruiting in C. neoformans. PMID:15643070

  20. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms.

    PubMed Central

    Neiman, A M; Stevenson, B J; Xu, H P; Sprague, G F; Herskowitz, I; Wigler, M; Marcus, S

    1993-01-01

    We present genetic evidence that three presumptive protein kinases of Schizosaccharomyces pombe, byr2, byr1, and spk1 that are structurally related to protein kinases of Saccharomyces cerevisiae, STE11, STE7, and FUS3, respectively, are also functionally related. In some cases, introduction of the heterologous protein kinase into a mutant was sufficient for complementation. In other cases (as in a ste11- mutant of S. cerevisiae), expression of two S. pombe protein kinases (byr2 and byr1) was required to observe complementation, suggesting that byr2 and byr1 act cooperatively. Complementation in S. pombe mutants is observed as restoration of sporulation and conjugation and in S. cerevisiae as restoration of conjugation, pheromone-induced cell cycle arrest, and pheromone-induced transcription of the FUS1 gene. We also show that the S. pombe kinases bear a similar relationship to the mating pheromone receptor apparatus as do their S. cerevisiae counterparts. Our results indicate that pheromone-induced signal transduction employs a conserved set of kinases in these two evolutionarily distant yeasts despite an apparently significant difference in function of the heterotrimeric G proteins. We suggest that the STE11/byr2, STE7/byr1, and FUS3/spk1 kinases comprise a signal transduction module that may be conserved in higher eukaryotes. Consistent with this hypothesis, we show that a mammalian mitogen-activated protein (MAP) kinase, ERK2, can partially replace spk1 function in S. pombe. Images PMID:8443406

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Preparation of Saccharomyces cerevisiae expression plasmids.

    PubMed

    Drew, David; Kim, Hyun

    2012-01-01

    Expression plasmids for Saccharomyces cerevisiae offer a wide choice of vector copy number, promoters of varying strength and selection markers. These expression plasmids are usually shuttle vectors that can be propagated both in yeast and bacteria, making them useful in gene cloning. For heterologous production of membrane proteins, we used the green fluorescent protein (GFP) fusion technology which was previously developed in the Escherichia coli system. We designed an expression plasmid carrying an inducible GAL1 promoter, a gene encoding a membrane protein of interest and the GFP-octa-histidine sequence. Here we describe construction of multi-copy yeast expression plasmids by homologous recombination in S. cerevisiae. PMID:22454112

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

    NASA Astrophysics Data System (ADS)

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

    2006-07-01

    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 15,000 membrane proteins from 38 fully sequenced eukaryotic genomes. membrane proteins | membrane proteomics | yeast

  8. Homology, Analogy, and Ethology.

    ERIC Educational Resources Information Center

    Beer, Colin G.

    1984-01-01

    Because the main criterion of structural homology (the principle of connections) does not exist for behavioral homology, the utility of the ethological concept of homology has been questioned. The confidence with which behavioral homologies can be claimed varies inversely with taxonomic distance. Thus, conjectures about long-range phylogenetic…

  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. Conservation of Histone Binding and Transcriptional Repressor Functions in a Schizosaccharomyces pombe Tup1p Homolog

    PubMed Central

    Mukai, Yukio; Matsuo, Eri; Roth, Sharon Y.; Harashima, Satoshi

    1999-01-01

    The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned from Kluyveromyces lactis (TUP1) and Schizosaccharomyces pombe (tup11+). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11+ gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, an S. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiae corresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11+ in combination with a disruption of tup12+, another TUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1+, suggesting that S. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species. PMID:10567571

  11. 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

  12. 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.

  13. 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

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...

  18. Braid Floer homology

    NASA Astrophysics Data System (ADS)

    van den Berg, J. B.; Ghrist, R.; Vandervorst, R. C.; Wójcik, W.

    2015-09-01

    Area-preserving diffeomorphisms of a 2-disc can be regarded as time-1 maps of (non-autonomous) Hamiltonian flows on R / Z ×D2. The periodic flow-lines define braid (conjugacy) classes, up to full twists. We examine the dynamics relative to such braid classes and define a new invariant for such classes, the BRAID FLOER HOMOLOGY. This refinement of Floer homology, originally used for the Arnol'd Conjecture, yields a Morse-type forcing theory for periodic points of area-preserving diffeomorphisms of the 2-disc based on braiding. Contributions of this paper include (1) a monotonicity lemma for the behavior of the nonlinear Cauchy-Riemann equations with respect to algebraic lengths of braids; (2) establishment of the topological invariance of the resulting braid Floer homology; (3) a shift theorem describing the effect of twisting braids in terms of shifting the braid Floer homology; (4) computation of examples; and (5) a forcing theorem for the dynamics of Hamiltonian disc maps based on braid Floer homology.

  19. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  20. The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila.

    PubMed Central

    Rong, Yikang S; Golic, Kent G

    2003-01-01

    In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining. PMID:14704169

  1. 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

  2. Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae.

    PubMed Central

    Jensen, M A; True, H L; Chernoff, Y O; Lindquist, S

    2001-01-01

    The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature. PMID:11606530

  3. 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

  4. DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

    PubMed Central

    Kuzminov, Andrei

    2001-01-01

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair. PMID:11459990

  5. 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),…

  6. Peptidase activities in Saccharomyces cerevisiae.

    PubMed Central

    Rose, B; Becker, J M; Naider, F

    1979-01-01

    At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives. Images PMID:378955

  7. 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

  8. Abelian link invariants and homology

    SciTech Connect

    Guadagnini, Enore; Mancarella, Francesco

    2010-06-15

    We consider the link invariants defined by the quantum Chern-Simons field theory with compact gauge group U(1) in a closed oriented 3-manifold M. The relation of the Abelian link invariants with the homology group of the complement of the links is discussed. We prove that, when M is a homology sphere or when a link--in a generic manifold M--is homologically trivial, the associated observables coincide with the observables of the sphere S{sup 3}. Finally, we show that the U(1) Reshetikhin-Turaev surgery invariant of the manifold M is not a function of the homology group only, nor a function of the homotopy type of M alone.

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

    PubMed

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

    2016-01-01

    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. PMID:27250940

  10. Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.

    PubMed

    Yu, Xin; Gabriel, Abram

    2004-02-01

    Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair. PMID:15020464

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

    PubMed

    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

  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. Characterization of RAD51-Independent Break-Induced Replication That Acts Preferentially with Short Homologous Sequences

    PubMed Central

    Ira, Grzegorz; Haber, James E.

    2002-01-01

    Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase. PMID:12192038

  14. 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

  15. 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

  16. 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

  17. Genetic variation of the repeated MAL loci in natural populations of Saccharomyces cerevisiae and Saccharomyces paradoxus.

    PubMed

    Naumov, G I; Naumova, E S; Michels, C A

    1994-03-01

    In Saccharomyces cerevisiae, the gene functions required to ferment the disaccharide maltose are encoded by the MAL loci. Any one of five highly sequence homologous MAL loci identified in various S. cerevisiae strains (called MAL1, 2, 3, 4 and 6) is sufficient to ferment maltose. Each is a complex of three genes encoding maltose permease, maltase and a transcription activator. This family of loci maps to telomere-linked positions on different chromosomes and most natural strains contain more than one MAL locus. A number of naturally occurring, mutant alleles of MAL1 and MAL3 have been characterized which lack one or more of the gene functions encoded by the fully functional MAL loci. Loss of these gene functions appears to have resulted from mutation and/or rearrangement within the locus. Studies to date concentrated on the standard maltose fermenting strains of S. cerevisiae available from the Berkeley Yeast Stock Center collection. In this report we extend our genetic analysis of the MAL loci to a number of maltose fermenting and nonfermenting natural strains of S. cerevisiae and Saccharomyces paradoxus. No new MAL loci were discovered but several new mutant alleles of MAL1 were identified. The evolution of this gene family is discussed. PMID:8005435

  18. 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.

  19. 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

  20. Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

    PubMed Central

    Risseeuw, E; Franke-van Dijk, M E; Hooykaas, P J

    1996-01-01

    Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector. PMID:8816506

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Methionine catabolism in Saccharomyces cerevisiae.

    PubMed

    Perpète, Philippe; Duthoit, Olivier; De Maeyer, Simon; Imray, Louise; Lawton, Andrew I; Stavropoulos, Konstantinos E; Gitonga, Virginia W; Hewlins, Michael J E; Dickinson, J Richard

    2006-01-01

    The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol. PMID:16423070

  9. 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. PMID:26205245

  10. 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

  11. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

    Resnick, M.A.; Boyce, J.; Cox, B.

    1981-04-01

    Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light. Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were chased, the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, it was concluded that postreplication repair in excision-defective mutants does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.

  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. 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

  14. Homologies in Physics and Astrophysics

    NASA Astrophysics Data System (ADS)

    Bartlett, David F.; Cumalat, J. P.

    2012-01-01

    The genes of humans and chimpanzees are homologs. These genes are - in large measure - identical. From this detailed observation, we naturally suppose that both species evolved from a common ancestor. In particle physics the ordinary observed particles and their superymmetric partners are thought to be homologs, generated by a common "ancestor” , the Higgs particle. Experiments at CERN currently are testing this comfortable analogy of physics with biology. Neither the Higgs boson nor any supersymmetric particle has yet been found. We speculate that a variety of objects are homologs - evidence of an as yet undeveloped quantum theory of gravity to replace Dark Matter. A purely astronomical homology is the Vc - σ o relation which places nearly spherical elliptical galaxies just above well-formed spirals (SA & SB). Here the asymptotically- flat, circular velocity Vc is observed to be between 1 and 2 times the central bulge velocity dispersion σo over the range 60 km/s< σo <400 km/s (Ferrarese 2002, Fig 3). The Vc - σ o relation is difficult to explain with self-consistent equilibrium galaxy models (Courteau et al 2007). Here we give an explanation based on the Sinusoidal Potential, a non-Newtonian potential in which φ =-GM Cos[ko r]/r and ko=2 π /400 pc. We relate the lower limit of 60 km/s to the thermal velocity of protons at the” Broadhurst/Hirano & Hartnett” lookback redshift Z=105.6. This is the redshift where what was 400 pc then expands to 128 h-1 Mpc today. Further, at this Z the temperature of the universe was close to the Hartree Energy of 2 times 13.6 eV, an energy where protons have an rms speed of about 60 km/s.

  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) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... controls). The special control is FDA's “Guidance for Industry and FDA Reviewers: Class II Special...

  16. Symplectic homology product via Legendrian surgery.

    PubMed

    Bourgeois, Frédéric; Ekholm, Tobias; Eliashberg, Yakov

    2011-05-17

    This research announcement continues the study of the symplectic homology of Weinstein manifolds undertaken by the authors [Bourgeois F, Ekholm T, Eliashberg Y (2009) arXiv:0911.0026] where the symplectic homology, as a vector space, was expressed in terms of the Legendrian homology algebra of the attaching spheres of critical handles. Here, we express the product and Batalin-Vilkovisky operator of symplectic homology in that context. PMID:21518898

  17. 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,

  18. Regulation of the Hansenula polymorpha maltase gene promoter in H. polymorpha and Saccharomyces cerevisiae1.

    PubMed

    Alamäe, Tiina; Pärn, Pille; Viigand, Katrin; Karp, Helen

    2003-11-01

    Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Delta failed to grow on maltose and sucrose, showing that maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Delta from the truncated variants of the promoter enabled us to define the 5'-upstream region as sufficient for the induction of maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Delta from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter. PMID:14613881

  19. Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.

    PubMed

    Viña-Gonzalez, Javier; Gonzalez-Perez, David; Alcalde, Miguel

    2016-01-01

    Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps. PMID:27077451

  20. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

    PubMed Central

    Fang, Fang; Salmon, Kirsty; Shen, Michael W. Y.; Aeling, Kimberly A.; Ito, Elaine; Irwin, Becky; Tran, Uyen Phuong C.; Hatfield, G. Wesley; Da Silva, Nancy A.; Sandmeyer, Suzanne

    2011-01-01

    A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 μ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering. PMID:20936606

  7. [Urinary infection by Saccharomyces cerevisiae: Emerging yeast?].

    PubMed

    Elkhihal, B; Elhalimi, M; Ghfir, B; Mostachi, A; Lyagoubi, M; Aoufi, S

    2015-12-01

    Saccharomyces cerevisiae is a commensal yeast of the digestive, respiratory and genito-urinary tract. It is widely used as a probiotic for the treatment of post-antibiotic diarrhea. It most often occurs in immunocompromised patients frequently causing fungemia. We report the case of an adult diabetic patient who had a urinary tract infection due to S. cerevisiae. The disease started with urination associated with urinary frequency burns without fever. The diagnosis was established by the presence of yeasts on direct examination and positivity of culture on Sabouraud-chloramphenicol three times. The auxanogramme gallery (Auxacolor BioRad(®)) allowed the identification of S. cerevisiae. The patient was put on fluconazole with good outcome. This observation points out that this is an opportunistic yeast in immunocompromised patients. PMID:26522963

  8. 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

  9. Investigation of Batten disease with the yeast Saccharomyces cerevisiae.

    PubMed

    Pearce, D A; Sherman, F

    1999-04-01

    The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease. PMID:10191120

  10. 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.

  11. 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.

  12. 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

  13. 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. PMID:26376869

  14. The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae.

    PubMed

    Brummer, M H; Richard, P; Sundqvist, L; Väänänen, R; Keränen, S

    2001-07-01

    The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. PMID:11447595

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

    PubMed

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

    2016-06-20

    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

  16. 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

  17. Chromosomally-retained RNA mediates homologous pairing.

    PubMed

    Ding, Da-Qiao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2012-01-01

    Pairing and recombination of homologous chromosomes are essential for ensuring correct segregation of chromosomes in meiosis. In S. pombe, chromosomes are first bundled at the telomeres (forming a telomere bouquet) and then aligned by oscillatory movement of the elongated "horsetail" nucleus. Telomere clustering and subsequent chromosome alignment promote pairing of homologous chromosomes. However, this telomere-bundled alignment of chromosomes cannot be responsible for the specificity of chromosome pairing. Thus, there must be some mechanism to facilitate recognition of homologous partners after telomere clustering. Recent studies in S. pombe have shown that RNA transcripts retained on the chromosome, or RNA bodies, may play a role in recognition of homologous chromosomes for pairing. Acting as fiducial markers of homologous loci they would abrogate the need for direct DNA sequence homology searching. PMID:23117617

  18. Asymmetrical division of Saccharomyces cerevisiae.

    PubMed Central

    Lord, P G; Wheals, A E

    1980-01-01

    The unequal division model proposed for budding yeast (L. H. Hartwell and M. W. Unger, J. Cell Biol. 75:422-435, 1977) was tested by bud scar analyses of steady-state exponential batch cultures of Saccharomyces cerevisiae growing at 30 degrees C at 19 different rates, which were obtained by altering the carbon source. The analyses involved counting the number of bud scars, determining the presence or absence of buds on at least 1,000 cells, and independently measuring the doubling times (gamma) by cell number increase. A number of assumptions in the model were tested and found to be in good agreement with the model. Maximum likelihood estimates of daughter cycle time (D), parent cycle time (P), and the budded phase (B) were obtained, and we concluded that asymmetrical division occurred at all growth rates tested (gamma, 75 to 250 min). D, P, and B are all linearly related to gamma, and D, P, and gamma converge to equality (symmetrical division) at gamma = 65 min. Expressions for the genealogical age distribution for asymmetrically dividing yeast cells were derived. The fraction of daughter cells in steady-state populations is e-alpha P, and the fraction of parent cells of age n (where n is the number of buds that a cell has produced) is (e-alpha P)n-1(1-e-alpha P)2, where alpha = IN2/gamma; thus, the distribution changes with growth rate. The frequency of cells with different numbers of bud scars (i.e., different genealogical ages) was determined for all growth rates, and the observed distribution changed with the growth rate in the manner predicted. In this haploid strain new buds formed adjacent to the previous buds in a regular pattern, but at slower growth rates the pattern was more irregular. The median volume of the cells and the volume at start in the cell cycle both increased at faster growth rates. The implications of these findings for the control of the cell cycle are discussed. PMID:6991494

  19. 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. PMID:26774271

  20. Homology-independent metrics for comparative genomics.

    PubMed

    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

  1. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant eff...

  2. Calcium control of Saccharomyces cerevisiae actin assembly.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo. Images PMID:6757718

  3. Biosorption of heavy metals by Saccharomyces cerevisiae.

    PubMed

    Volesky, B; May-Phillips, H A

    1995-01-01

    Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4-5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu approximately (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast (approximately 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine needle-like crystals both on the inside and outside of the S. cerevisiae cells. PMID:7765919

  4. 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…

  5. Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22.

    PubMed Central

    Ono, Y; Ohno, M; Shimura, Y

    1994-01-01

    In the budding yeast Saccharomyces cerevisiae, a number of PRP genes known to be involved in pre-mRNA processing have been genetically identified and cloned. Three PRP genes (PRP2, PRP16, and PRP22) were shown to encode putative RNA helicases of the family of proteins with DEAH boxes. However, any such splicing factor containing the helicase motifs in vertebrates has not been identified. To identify human homologs of this family, we designed PCR primers corresponding to the highly conserved region of the DEAH box protein family and successfully amplified five cDNA fragments, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which was previously shown to be involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in a S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, HRH1 but not Prp22 contains an arginine- and serine-rich domain (RS domain) which is characteristic of some splicing factors, such as members of the SR protein family. We could show that HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. We speculate that HRH1 might be targeted to the spliceosome through this interaction. Images PMID:7935475

  6. 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).

  7. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  8. Gene Sequence Homology of Chemokines Across Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The abundance of expressed gene and protein sequences available in the biological information databases facilitates comparison of protein homologies. A high degree of sequence similarity typically implies homology regarding structure and function and may provide clues to antibody cross-reactivities...

  9. GENE SEQUENCE HOMOLOGY OF CHEMOKINES ACROSS SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The abundance of expressed gene and protein sequences available in the biological information databases facilitates comparison of protein homologies. A high degree of sequence similarity typically implies homology regarding structure and function and may provide clues to antibody cross-react...

  10. Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

    PubMed Central

    Symington, Lorraine S.

    2002-01-01

    The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination. PMID:12456786

  11. 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.

  12. Sporulation in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Neiman, Aaron M.

    2011-01-01

    In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423

  13. 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

  14. 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.

  15. Buoyancy instability of homologous implosions

    DOE PAGESBeta

    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

  16. 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

  17. 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

  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. Myo-inositol transport in Saccharomyces cerevisiae.

    PubMed

    Nikawa, J; Nagumo, T; Yamashita, S

    1982-05-01

    myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide. PMID:7040334

  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. A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

    PubMed

    Cuperus, Josh T; Lo, Russell S; Shumaker, Lucia; Proctor, Julia; Fields, Stanley

    2015-07-17

    Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes. PMID:25742460

  2. 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

  3. 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

  4. 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

  5. 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

  6. New phosphonate reagents for aldehyde homologation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New phosphonate reagents were developed for the two-carbon homologation of aldehydes to unbranched- or methyl-branched unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a protected...

  7. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

    PubMed Central

    Kruckeberg, A L; Bisson, L F

    1990-01-01

    The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes. Images PMID:2233722

  8. Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

    PubMed Central

    Srivatsan, Anjana; Bowen, Nikki; Gries, Kerstin; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

    2014-01-01

    In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1. PMID:24811092

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

    PubMed Central

    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

  10. 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.

  11. 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.

  12. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein.

    PubMed Central

    Celenza, J L; Marshall-Carlson, L; Carlson, M

    1988-01-01

    The SNF3 gene is required for high-affinity glucose transport in the yeast Saccharomyces cerevisiae and has also been implicated in control of gene expression by glucose repression. We report here the nucleotide sequence of the cloned SNF3 gene. The predicted amino acid sequence shows that SNF3 encodes a 97-kilodalton protein that is homologous to mammalian glucose transporters and has 12 putative membrane-spanning regions. We also show that a functional SNF3-lacZ gene-fusion product cofractionates with membrane proteins and is localized to the cell surface, as judged by indirect immunofluorescence microscopy. Expression of the fusion protein is regulated by glucose repression. Images PMID:3281163

  13. 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

  14. On the hodological criterion for homology.

    PubMed

    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. A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast.

    PubMed

    Brennwald, P; Wise, J A

    1994-02-01

    We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-alpha-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism. PMID:8203158

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

    PubMed

    Keyamura, Kenji; Arai, Kota; Hishida, Takashi

    2016-07-01

    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

  17. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    NASA Astrophysics Data System (ADS)

    Raffar, Nur Izzati Abdul; Rahman, Nadhratul Nur Ain Abdul; Alrozi, Rasyidah; Senusi, Faraziehan; Chang, Siu Hua

    2015-05-01

    Biosorption of copper ion using treated and untreated immobilized Saccharomyces cerevisiae from aqueous solution was investigate in this study. S.cerevisiae has been choosing as biosorbent due to low cost, easy and continuously available from various industries. In this study, the ability of treated and untreated immobilized S.cerevisiae in removing copper ion influence by the effect of pH solution, and initial concentration of copper ion with contact time. Besides, adsorption isotherm and kinetic model also studied. The result indicated that the copper ion uptake on treated and untreated immobilized S.cerevisiae was increased with increasing of contact time and initial concentration of copper ion. The optimum pH for copper ion uptake on untreated and treated immobilized S.cerevisiae at 4 and 6. From the data obtained of copper ion uptake, the adsorption isotherm was fitted well by Freundlich model for treated immobilized S.cerevisiae and Langmuir model for untreated immobilized S.cerevisiae according to high correlation coefficient. Meanwhile, the pseudo second order was described as suitable model present according to high correlation coefficient. Since the application of biosorption process has been received more attention from numerous researchers as a potential process to be applied in the industry, future study will be conducted to investigate the potential of immobilized S.cerevisiae in continuous process.

  18. Investigating homology between proteins using energetic profiles.

    PubMed

    Wrabl, James O; Hilser, Vincent J

    2010-03-01

    Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

  19. Homologs of Breast Cancer Genes in Plants

    PubMed Central

    Trapp, Oliver; Seeliger, Katharina; Puchta, Holger

    2011-01-01

    Since the initial discovery of genes involved in hereditary breast cancer in humans, a vast wealth of information has been published. Breast cancer proteins were shown to work as tumor suppressors primarily through their involvement in DNA-damage repair. Surprisingly, homologs of these genes can be found in plant genomes, as well. Here, we want to give an overview of the identification and characterization of the biological roles of these proteins, in plants. In addition to the conservation of their function in DNA repair, new plant-specific characteristics have been revealed. BRCA1 is required for the efficient repair of double strand breaks (DSB) by homologous recombination in somatic cells of the model plant Arabidopsis thaliana. Bioinformatic analysis indicates that, whereas most homologs of key components of the different mammalian BRCA1 complexes are present in plant genomes, homologs of most factors involved in the recruitment of BRCA1 to the DSB cannot be identified. Thus, it is not clear at the moment whether differences exist between plants and animals at this important step. The most conserved region of BRCA1 and BARD1 homologs in plants is a PHD domain which is absent in mammals and which, in AtBARD1, might be involved in the transcriptional regulation of plant development. The presence of a plant-specific domain prompted us to reevaluate the current model for the evolution of BRCA1 homologs and to suggest a new hypothesis, in which we postulate that plant BRCA1 and BARD1 have one common predecessor that gained a PHD domain before duplication. Furthermore, work in Arabidopsis demonstrates that – as in animals – BRCA2 homologs are important for meiotic DNA recombination. Surprisingly, recent research has revealed that AtBRCA2 also has an important role in systemic acquired resistance. In Arabidopsis, BRCA2 is involved in the transcriptional regulation of pathogenesis-related (PR) genes via its interaction with the strand exchange protein RAD51. PMID

  20. A G-protein alpha subunit from asexual Candida albicans functions in the mating signal transduction pathway of Saccharomyces cerevisiae and is regulated by the a1-alpha 2 repressor.

    PubMed Central

    Sadhu, C; Hoekstra, D; McEachern, M J; Reed, S I; Hicks, J B

    1992-01-01

    We have isolated a gene, designated CAG1, from Candida albicans by using the G-protein alpha-subunit clone SCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is more homologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not only includes the conserved guanine nucleotide binding domains but also spans the normally variable regions which are thought to be involved in interaction with the components of the specific signal transduction pathway. Furthermore, CAG1 contains a central domain, previously found only in SCG1. cag1 null mutants of C. albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAG1 gene complemented both the growth and mating defects of S. cerevisiae scg1 null mutants when carried on either a low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable in mycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S. cerevisiae transformants containing the C. albicans CAG1 gene was observed only in haploid cells. This transcription pattern matches that of SCG1 in S. cerevisiae and is caused by a1-alpha 2 mediated repression in diploid cells. That is, CAG1 behaves as a haploid-specific gene in S. cerevisiae, subject to control by the a1-alpha 2 mating-type regulation pathway. We infer from these results that C. albicans may have a signal transduction system analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so far remained undetected. Images PMID:1569935

  1. [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

  2. 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

  3. 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

  4. 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

  5. Mycobacterium tuberculosis expresses two chaperonin-60 homologs.

    PubMed Central

    Kong, T H; Coates, A R; Butcher, P D; Hickman, C J; Shinnick, T M

    1993-01-01

    A 65-kDa protein and a 10-kDa protein are two of the more strongly immunoreactive components of Mycobacterium tuberculosis, the causative agent of tuberculosis. The 65-kDa antigen has homology with members of the GroEL or chaperonin-60 (Cpn60) family of heat shock proteins. The 10-kDa antigen has homology with the GroES or chaperonin-10 family of heat shock proteins. These two proteins are encoded by separate genes in M. tuberculosis. The studies reported here reveal that M. tuberculosis contains a second Cpn60 homolog located 98 bp downstream of the 10-kDa antigen gene. The second Cpn60 homolog (Cpn60-1) displays 61% amino acid sequence identity with the 65-kDa antigen (Cpn60-2) and 53% and 41% identity with the Escherichia coli GroEL protein and the human P60 protein, respectively. Primer-extension analysis revealed that transcription starts 29 bp upstream of the translation start of the Cpn60-1 homolog and protein purification studies indicate that the cpn60-1 gene is expressed as an approximately 60-kDa polypeptide. Images Fig. 3 Fig. 5 PMID:7681982

  6. 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.

  7. 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.

  8. Flare build-up study: Homologous flares group - Interim report

    NASA Technical Reports Server (NTRS)

    Woodgate, B. E.

    1982-01-01

    When homologous flares are broadly defined as having footpoint structures in common, it is found that a majority of flares fall into homologous sets. Filament eruptions and mass ejection in members of an homologous flare set show that maintainance of the magnetic structure is not a necessary condition for homology.

  9. Cloning of the STE5 gene of Saccharomyces cerevisiae as a suppressor of the mating defect of cdc25 temperature-sensitive mutants.

    PubMed Central

    Perlman, R; Yablonski, D; Simchen, G; Levitzki, A

    1993-01-01

    The STE5 gene of Saccharomyces cerevisiae was cloned using a screening procedure designed to isolate genes of the S. cerevisiae pheromone response pathway. We screened a yeast genomic high-copy-number plasmid library for genes that allow mating of cdc25ts mutants at the restrictive temperature without affecting the cell-cycle-arrest phenotype. One of the genes cloned was identified by genetic analysis as STE5. STE5 encodes a predicted open reading frame of 916 amino acids and exhibits significant homology to Far1 protein. RNA blot analysis reveals that STE5 gene transcription is regulated by the mating type of the cell and depends on an intact pheromone-response pathway. Images Fig. 1 Fig. 5 PMID:8516289

  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. 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

  12. 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

  13. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52.

    PubMed

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  14. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52

    PubMed Central

    Di Primio, Cristina; Galli, Alvaro; Cervelli, Tiziana; Zoppè, Monica; Rainaldi, Giuseppe

    2005-01-01

    When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies. PMID:16106043

  15. 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. PMID:27214570

  16. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    PubMed

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  17. Genome-Wide Screen Reveals Replication Pathway for Quasi-Palindrome Fragility Dependent on Homologous Recombination

    PubMed Central

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S.

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  18. 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

  19. A homolog of lariat-debranching enzyme modulates turnover of branched RNA

    PubMed Central

    Garrey, Stephen M.; Katolik, Adam; Prekeris, Mantas; Li, Xueni; York, Kerri; Bernards, Sarah; Fields, Stanley; Zhao, Rui; Damha, Masad J.; Hesselberth, Jay R.

    2014-01-01

    Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudo-metallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5′-exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing. PMID:24919400

  20. 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.

  1. Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination.

    PubMed

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  2. 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

  3. 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

  4. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    PubMed

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis. PMID:26254786

  5. Metazoan Scc4 Homologs Link Sister Chromatid Cohesion to Cell and Axon Migration Guidance

    PubMed Central

    Seitan, Vlad C; Banks, Peter; Laval, Steve; Majid, Nazia A; Dorsett, Dale; Rana, Amer; Smith, Jim; Bateman, Alex; Krpic, Sanja; Hostert, Arnd; Rollins, Robert A; Erdjument-Bromage, Hediye; Tempst, Paul; Benard, Claire Y; Hekimi, Siegfried; Newbury, Sarah F

    2006-01-01

    Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development. PMID:16802858

  6. Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance.

    PubMed

    Seitan, Vlad C; Banks, Peter; Laval, Steve; Majid, Nazia A; Dorsett, Dale; Rana, Amer; Smith, Jim; Bateman, Alex; Krpic, Sanja; Hostert, Arnd; Rollins, Robert A; Erdjument-Bromage, Hediye; Tempst, Paul; Benard, Claire Y; Hekimi, Siegfried; Newbury, Sarah F; Strachan, Tom

    2006-07-01

    Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein-protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development. PMID:16802858

  7. 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. PMID:26519319

  8. 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.

  9. 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

  10. Invasive Saccharomyces cerevisiae infection: a friend turning foe?

    PubMed

    Pillai, Unnikrishnan; Devasahayam, Joe; Kurup, Aparna Narayana; Lacasse, Alexandre

    2014-11-01

    We report a very rare case of acute pyelonephritis in a 51-year-old female with a history of chronic kidney disease (CKD) and diabetes caused by a normally benign and a well-known human commensal organism, Saccharomyces cerevisiae that is very often prescribed as a probiotic in modern medical practice. The causal role of S. cerevisiae was confirmed by its isolation in blood, urine, stool as well as vaginal swabs thus proving its virulent nature in suitable situations. PMID:25394448

  11. A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

    PubMed

    Aouida, Mustapha; Kim, Kangchang; Shaikh, Abdul Rajjak; Pardo, Jose M; Eppinger, Jörg; Yun, Dae-Jin; Bressan, Ray A; Narasimhan, Meena L

    2013-01-01

    Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to

  12. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  13. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    PubMed Central

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  14. 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.

  15. 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

  16. Homologous Pairing between Long DNA Double Helices.

    PubMed

    Mazur, Alexey K

    2016-04-15

    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. PMID:27127987

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  2. 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.

  3. Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor, from Yeast Mating to Human Disease

    PubMed Central

    Barrowman, Jemima

    2012-01-01

    Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery. PMID:22933563

  4. Size threshold for Saccharomyces cerevisiae chromosomes: generation of telocentric chromosomes from an unstable minichromosome.

    PubMed Central

    Zakian, V A; Blanton, H M; Wetzel, L; Dani, G M

    1986-01-01

    A 9-kilobase pair CEN4 linear minichromosome constructed in vitro transformed Saccharomyces cerevisiae with high frequency but duplicated or segregated inefficiently in most cells. Stable transformants were only produced by events which fundamentally altered the structure of the minichromosome: elimination of telomeres, alteration of the centromere, or an increase of fivefold or greater in its size. Half of the stable transformants arose via homologous recombination between an intact chromosome IV and the CEN4 minichromosome. This event generated a new chromosome from each arm of chromosome IV. The other "arm" of each new chromosome was identical to one "arm" of the unstable minichromosome. Unlike natural yeast chromosomes, these new chromosomes were telocentric: their centromeres were either 3.9 or 5.4 kilobases from one end of the chromosome. The mitotic stability of the telocentric chromosome derived from the right arm of chromosome IV was determined by a visual assay and found to be comparable to that of natural yeast chromosomes. Both new chromosomes duplicated, paired, and segregated properly in meiosis. Moreover, their structure, as deduced from mobilities in orthogonal field gels, did not change with continued mitotic growth or after passage through meiosis, indicating that they did not give rise to isochromosomes or suffer large deletions or additions. Thus, in S. cerevisiae the close spacing of centromeres and telomeres on a DNA molecule of chromosomal size does not markedly alter the efficiency with which it is maintained. Taken together these data suggest that there is a size threshold below which stable propagation of linear chromosomes is no longer possible. Images PMID:3022139

  5. 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

    2015-01-01

    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. PMID:26715090

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

    PubMed Central

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

    2015-01-01

    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. PMID:26715090

  7. 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

  8. 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

  9. 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

  10. 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

  11. A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

    PubMed Central

    Edwards, H; Schimmel, P

    1990-01-01

    Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA. Images PMID:1690848

  12. Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.

    PubMed Central

    Shaw, K J; Olson, M V

    1984-01-01

    Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expression; deletion mutants extending to positions -18, -17, -16, or -15 are moderately deficient; deletion mutants extending to positions -36 or -27 are slightly defective; and mutants retaining more than 60 base pairs of the original 5'-flanking DNA are expressed normally. In some cases, the cloning procedure led to the introduction of multiple BamHI linkers at the SUP4-o-vector fusion site, and in one instance, the resulting structure dramatically affects gene function: the presence of three linkers abutting a -18 deletion completely inhibits the in vivo expression of SUP4-o. In contrast, three linkers that abut a -77 deletion have no effect on in vivo expression. The template properties of these plasmids in a homologous in vitro transcription system parallel the levels of in vivo expression, suggesting that the mutations predominantly affect transcription. The data demonstrate that there are significant functional constraints on the 5'-flanking sequences of this RNA polymerase III-transcribed gene. The dramatic effects of the multiple linker insertion at position -18 suggest that there may be extensive melting of the DNA in this region during normal transcription initiation. Images PMID:6371493

  13. 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

  14. RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

    PubMed

    Lin, Qiuhui; Jia, Bin; Mitchell, Leslie A; Luo, Jingchuan; Yang, Kun; Zeller, Karen I; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S; Boeke, Jef D; Yuan, Ying-Jin

    2015-03-20

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects. PMID:24895839

  15. HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain.

    PubMed

    Kalchman, M A; Koide, H B; McCutcheon, K; Graham, R K; Nichol, K; Nishiyama, K; Kazemi-Esfarjani, P; Lynn, F C; Wellington, C; Metzler, M; Goldberg, Y P; Kanazawa, I; Gietz, R D; Hayden, M R

    1997-05-01

    Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain. PMID:9140394

  16. Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae

    PubMed Central

    Castle, Christopher D.; Sardana, Richa; Dandekar, Varada; Borgianini, Victoria; Johnson, Arlen W.; Denicourt, Catherine

    2013-01-01

    Ribosome biogenesis is a multi-step process that couples cell growth with cell proliferation. Although several large-scale analysis of pre-ribosomal particles have identified numerous trans-acting factors involved in this process, many proteins involved in pre-rRNA processing and ribosomal subunit maturation have yet to be identified. Las1 was originally identified in Saccharomyces cerevisiae as a protein involved in cell morphogenesis. We previously demonstrated that the human homolog, Las1L, is required for efficient ITS2 rRNA processing and synthesis of the 60S ribosomal subunit. Here, we report that the functions of Las1 in ribosome biogenesis are also conserved in S. cerevisiae. Depletion of Las1 led to the accumulation of both the 27S and 7S rRNA intermediates and impaired the synthesis of the 60S subunit. We show that Las1 co-precipitates mainly with the 27S rRNA and associates with an Nsa1 and Rix1-containing pre-60S particle. We further identify Grc3 as a major Las1-interacting protein. We demonstrate that the kinase activity of Grc3 is required for efficient pre-rRNA processing and that depletion of Grc3 leads to rRNA processing defects similar to the ones observed in Las1-depleted cells. We propose that Las1 and Grc3 function together in a conserved mechanism to modulate rRNA processing and eukaryotic ribosome biogenesis. PMID:23175604

  17. 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

  18. Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae.

    PubMed

    Warit, S; Walmsley, R M; Stateva, L I

    1998-09-01

    Two genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated. PMID:9782489

  19. Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

    PubMed Central

    2011-01-01

    We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete. PMID:21982381

  20. The Snf2 Homolog Fun30 Acts as a Homodimeric ATP-dependent Chromatin-remodeling Enzyme*

    PubMed Central

    Awad, Salma; Ryan, Daniel; Prochasson, Philippe; Owen-Hughes, Tom; Hassan, Ahmed H.

    2010-01-01

    The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones. PMID:20075079

  1. Studies of pH regulation by Btn1p, the yeast homolog of human Cln3p.

    PubMed

    Pearce, D A; Nosel, S A; Sherman, F

    1999-04-01

    Although the gene responsible for Batten disease, CLN3, was positionally cloned in 1995, the function of Cln3p and the molecular basis of the disease still remain elusive. We previously reported that the yeast Saccharomyces cerevisiae contains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1, 3-propanediol in btn1-Delta yeast mutants. We have determined that yeast lacking Btn1p have an elevated ability to acidify media during growth that correlates with an elevated plasma membrane ATPase activity. Btn1p may be involved in maintaining pH homeostasis of yeast cells. PMID:10191121

  2. Regulation of Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Waechter, Charles J.; Lester, Robert L.

    1971-01-01

    Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate 14C-CH3-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of 14C-CH3-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium. Images PMID:5547992

  3. Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Culbertson, Michael R.; Henry, Susan A.

    1975-01-01

    Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974). PMID:1093935

  4. 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

  5. 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

  6. Homologous Series of Liquid Crystalline Steroidal Lipids

    SciTech Connect

    Thiemann, T.; Vill, V.

    1997-03-01

    Steroids are an important source of chiral mesophases. The melting behavior and mesomorphic properties of homologous series of steroidal derivatives have been extracted from the literature, tabulated, and discussed. The tables provide the reader with an evaluated compilation of the type of mesophases found for the individual compounds, including their transition temperatures. Where the literature gives more than one set of data for a specific substance, one has been chosen as the main reference, but all alternatives are listed in the footnotes. The data can be used for statistical analysis to show the specific role of substructures within the steroidal framework. {copyright} {ital 1997} {ital American Institute of Physics and American Chemical Society}

  7. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

    PubMed

    van der Spek, P J; Visser, C E; Hanaoka, F; Smit, B; Hagemeijer, A; Bootsma, D; Hoeijmakers, J H

    1996-01-01

    The Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone repair syndrome xeroderma pigmentosum, complementation group C, and is specifically involved in the global genome NER subpathway. The cloning of both mouse homologs (designated MHR23A and MHR23B) and detailed sequence comparison permitted the deduction of the following overall structure for all RAD23 homologs: an ubiquitin-like N-terminus followed by a strongly conserved 50-amino-acid domain that is repeated at the C-terminus. We also found this domain as a specific C-terminal extension of one of the ubiquitin-conjugating enzymes, providing a second link with the ubiquitin pathway. By means of in situ hybridization, MHR23A was assigned to mouse chromosome 8C3 and MHR23B to 4B3. Because of the close chromosomal proximity of human XPC and HHR23B, the mouse XPC chromosomal location was determined (6D). Physical disconnection of the genes in mouse argues against a functional significance of the colocalization of these genes in human. Northern blot analysis revealed constitutive expression of both MHR23 genes in all tissues examined. Elevated RNA expression of both MHR23 genes was observed in testis. Although the RAD23 equivalents are well conserved during evolution, the mammalian genes did not express the UV-inducible phenotype of their yeast counterpart. This may point to a fundamental difference between the UV responses of yeast and human. No stage-specific mRNA expression during the cell cycle was observed for the mammalian RAD23 homologs. PMID:8808275

  8. Towards Alignment Independent Quantitative Assessment of Homology Detection

    PubMed Central

    Kliger, Yossef

    2006-01-01

    Identification of homologous proteins provides a basis for protein annotation. Sequence alignment tools reliably identify homologs sharing high sequence similarity. However, identification of homologs that share low sequence similarity remains a challenge. Lowering the cutoff value could enable the identification of diverged homologs, but also introduces numerous false hits. Methods are being continuously developed to minimize this problem. Estimation of the fraction of homologs in a set of protein alignments can help in the assessment and development of such methods, and provides the users with intuitive quantitative assessment of protein alignment results. Herein, we present a computational approach that estimates the amount of homologs in a set of protein pairs. The method requires a prevalent and detectable protein feature that is conserved between homologs. By analyzing the feature prevalence in a set of pairwise protein alignments, the method can estimate the number of homolog pairs in the set independently of the alignments' quality. Using the HomoloGene database as a standard of truth, we implemented this approach in a proteome-wide analysis. The results revealed that this approach, which is independent of the alignments themselves, works well for estimating the number of homologous proteins in a wide range of homology values. In summary, the presented method can accompany homology searches and method development, provides validation to search results, and allows tuning of tools and methods. PMID:17205117

  9. 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

  10. Saccharomyces cerevisiae: a nomadic yeast with no niche?

    PubMed Central

    Goddard, Matthew R.; Greig, Duncan

    2015-01-01

    Different species are usually thought to have specific adaptations, which allow them to occupy different ecological niches. But recent neutral ecology theory suggests that species diversity can simply be the result of random sampling, due to finite population sizes and limited dispersal. Neutral models predict that species are not necessarily adapted to specific niches, but are functionally equivalent across a range of habitats. Here, we evaluate the ecology of Saccharomyces cerevisiae, one of the most important microbial species in human history. The artificial collection, concentration and fermentation of large volumes of fruit for alcohol production produce an environment in which S. cerevisiae thrives, and therefore it is assumed that fruit is the ecological niche that S. cerevisiae inhabits and has adapted to. We find very little direct evidence that S. cerevisiae is adapted to fruit, or indeed to any other specific niche. We propose instead a neutral nomad model for S. cerevisiae, which we believe should be used as the starting hypothesis in attempting to unravel the ecology of this important microbe. PMID:25725024

  11. Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.

    PubMed

    Mormeneo, María; Pastor, Fi Javier; Zueco, Jesús

    2012-01-01

    The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase. PMID:21701899

  12. 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

  13. Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH.

    PubMed Central

    Hansen, J; Cherest, H; Kielland-Brandt, M C

    1994-01-01

    The yeast assimilatory sulfate reductase is a complex enzyme that is responsible for conversion of sulfite into sulfide. To obtain information on the nature of this enzyme, we isolated and sequenced the MET10 gene of Saccharomyces cerevisiae and a divergent MET10 allele from Saccharomyces carlsbergensis. The polypeptides deduced from the identically sized open reading frames (1,035 amino acids) of both MET10 genes have molecular masses of around 115 kDa and are 88% identical to each other. The transcript of S. cerevisiae MET10 has a size comparable to that of the open reading frame and is transcriptionally repressed by methionine in a way similar to that seen for other MET genes of S. cerevisiae. Distinct homology was found between the putative MET10-encoded polypeptide and flavin-interacting parts of the sulfite reductase flavoprotein subunit (encoded by cysJ) from Escherichia coli and several other flavoproteins. A significant N-terminal homology to pyruvate flavodoxin oxidoreductase (encoded by nifJ) from Klebsiella pneumoniae, together with a lack of obvious flavin mononucleotide-binding motifs in the MET10 deduced amino acid sequence, suggests that the yeast assimilatory sulfite reductase is a distinct type of sulfite reductase. Images PMID:7928966

  14. 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

  15. 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.

  16. 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. PMID:26517909

  17. SANSparallel: interactive homology search against Uniprot

    PubMed Central

    Somervuo, Panu; Holm, Liisa

    2015-01-01

    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

  18. 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

  19. 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.

  20. 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. PMID:25593311

  1. 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

  2. Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

    PubMed

    Eksteen, Jeremy M; Steyn, Andries J C; van Rensburg, Pierre; Cordero Otero, Ricardo R; Pretorius, Isak S

    2003-01-15

    Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha

  3. 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

  4. Prediction of Saccharomyces cerevisiae replication origins

    PubMed Central

    Breier, Adam M; Chatterji, Sourav; Cozzarelli, Nicholas R

    2004-01-01

    Background Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. Results We developed an algorithm, Oriscan, to predict yeast origins using similarity to 26 characterized origins. Oriscan used 268 bp of sequence, including the T-rich ACS and a 3' A-rich region. The predictions identified the exact location of the ACS. A total of 84 of the top 100 Oriscan predictions, and 56% of the top 350, matched known ARSs or replication protein binding sites. The true accuracy was even higher because we tested 25 discrepancies, and 15 were in fact ARSs. Thus, 94% of the top 100 predictions and an estimated 70% of the top 350 were correct. We compared the predictions to corresponding sequences in related Saccharomyces species and found that the ACSs of experimentally supported predictions show significant conservation. Conclusions The high accuracy of the predictions indicates that we have defined near-sufficient conditions for ARS activity, the A-rich region is a recognizable feature of ARS elements with a probable role in replication initiation, and nucleotide sequence is a reliable predictor of yeast origins. Oriscan detected most origins in the genome, demonstrating previously unrecognized generality in yeast replication origins and significant discriminatory power in the algorithm. PMID:15059255

  5. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-11-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  6. 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

  7. 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

  8. 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

  9. Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas

    PubMed Central

    King, Nichole L; Deutsch, Eric W; Ranish, Jeffrey A; Nesvizhskii, Alexey I; Eddes, James S; Mallick, Parag; Eng, Jimmy; Desiere, Frank; Flory, Mark; Martin, Daniel B; Kim, Bong; Lee, Hookeun; Raught, Brian; Aebersold, Ruedi

    2006-01-01

    We present the Saccharomyces cerevisiae PeptideAtlas composed from 47 diverse experiments and 4.9 million tandem mass spectra. The observed peptides align to 61% of Saccharomyces Genome Database (SGD) open reading frames (ORFs), 49% of the uncharacterized SGD ORFs, 54% of S. cerevisiae ORFs with a Gene Ontology annotation of 'molecular function unknown', and 76% of ORFs with Gene names. We highlight the use of this resource for data mining, construction of high quality lists for targeted proteomics, validation of proteins, and software development. PMID:17101051

  10. A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements.

    PubMed

    Moskvina, E; Schüller, C; Maurer, C T; Mager, W H; Ruis, H

    1998-08-01

    Stress response elements (STREs, core consensus AG4 or C4T) have been demonstrated previously to occur in the upstream region of a number of genes responsive to induction by a variety of stress signals. This stress response is mediated by the homologous transcription factors Msn2p and Msn4p, which bind specifically to STREs. Double mutants (msn2 msn4) deficient in these transcription factors have been shown to be hypersensitive to severe stress conditions. To obtain a more representative overview of the set of yeast genes controlled via this regulon, a computer search of the Saccharomyces cerevisiae genome was carried out for genes, which, similar to most known STRE-controlled genes, exhibit at least two STREs in their upstream region. In addition to the great majority of genes previously known to be controlled via STREs, 69 open reading-frames were detected. Expression patterns of a set of these were examined by grid filter hybridization, and 14 genes were examined by Northern analysis. Comparison of the expression patterns of these genes demonstrates that they are all STRE-controlled although their detailed expression patterns differ considerably. PMID:9730283